Immobilization on a flow cell

Abstract

In one example, a target material is immobilized on two opposing sequencing surfaces of a flow cell using a first and a second fluid. The first fluid has a density lower than that of the target material and the second fluid has a density higher than that of the target material, or the second fluid has a density lower than that of the target material and the first fluid has a density higher than that of the target material. The first fluid (containing the target material) is introduced into the flow cell, causing at least a portion of the target material to be immobilized by capture sites on one of the sequencing surfaces. The first fluid and any unimmobilized target material are removed. The second fluid (containing the target material) is introduced into the flow cell, causing at least a portion of the target material to be immobilized by capture sites on the other sequencing surface.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 946,717, filed December 11, 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Flow cells are used in a variety of methods and applications, including gene sequencing and genotyping. In some methods and applications, it is desirable to generate a library of fragmented and tagged DNA molecules from a double-stranded DNA (dsDNA) target molecule. Often, the goal is to generate smaller DNA molecules (e.g., DNA fragments) from larger dsDNA molecules for use as templates in DNA sequencing reactions. The templates can allow for short read lengths to be obtained. During data analysis, overlapping short sequence reads can be aligned to reconstruct a longer nucleic acid sequence. In some cases, a pre-sequencing step (e.g., barcoding specific nucleic acid molecules) can be used to simplify data analysis. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Provisional Patent Application No. 62 / 946,717 Summary of the Invention [Problem to be solved by the invention]

[0004] Some example kits and methods described herein are suitable for immobilizing one or more target materials on opposing surfaces of a flow cell, with some example methods allowing for sequential immobilization and others allowing for simultaneous immobilization. [Means for solving the problem]

[0005] A first aspect disclosed herein is a method comprising immobilizing a target material on each of two opposing sequencing surfaces of a flow cell, wherein the immobilization comprises introducing a first fluid into the flow cell, the first fluid having a first portion of the target material therein, thereby immobilizing at least a portion of the target material through capture sites on one of the opposing sequencing surfaces; removing the first fluid and any unimmobilized target material from the flow cell; and introducing a second fluid into the flow cell, the second fluid having a second portion of the target material therein, thereby immobilizing at least a portion of the target material through capture sites on the other of the opposing sequencing surfaces, wherein either the first fluid has a density lower than that of the target material and the second fluid has a density higher than that of the target material, or the second fluid has a density lower than that of the target material and the first fluid has a density higher than that of the target material.

[0006] A second aspect disclosed herein is a kit including preparation fluids having a target material therein, wherein a first introduction fluid has a density lower than that of the target material, and a second introduction fluid has a density higher than that of the target material.

[0007] A third aspect disclosed herein is a method comprising immobilizing target materials on each of two opposing sequencing surfaces of a flow cell by the steps of: introducing a fluid containing the target materials into the flow cell, the target materials including magnetic solid supports, sequencing-ready nucleic acid fragments or template strands attached to the magnetic solid supports, and the fluid having a density at least approximately equal to that of the magnetic solid supports; immobilizing some of the target materials by capture sites on one of the opposing sequencing surfaces; and applying a magnetic force to the other of the opposing sequencing surfaces, thereby pulling some other target materials onto the other of the opposing sequencing surfaces, whereby the target materials are immobilized by the capture sites on the other of the opposing sequencing surfaces.

[0008] A fourth aspect disclosed herein is a method comprising introducing a target fluid comprising a first target material and a second target material into a flow cell, thereby simultaneously immobilizing a first target material on a first surface of two opposing sequencing surfaces of the flow cell and a second target material on a second surface of two opposing sequencing surfaces, wherein a carrier fluid of the target fluid has a fluid density, the first target material has a first density less than the fluid density, and the second target material has a second density greater than the fluid density.

[0009] A fifth aspect disclosed herein is a target material including a carrier fluid having a fluid density, a first target material having a first density less than the fluid density, and a second target material having a second density greater than the fluid density.

[0010] A sixth aspect disclosed herein is a method comprising introducing first and second target materials into a flow cell comprising two opposing sequencing surfaces, wherein the first target material has at least one property that differs from the second target material, the at least one property being selected from the group consisting of density, charge, magnetism, and combinations thereof; and exposing the first and second target materials to at least one condition, whereby the first target material is immobilized by first capture sites on the two opposing sequencing surfaces and the second target material is immobilized by second capture sites on the two opposing sequencing surfaces.

[0011] It should be understood that any feature of any one of the embodiments can be combined together in any desired manner. Furthermore, it should be understood that any combination of the features of the first embodiment and / or the second embodiment and / or the third embodiment and / or the fourth embodiment and / or the fifth embodiment and / or the sixth embodiment can also be combined with any of the embodiments disclosed herein to achieve the benefits described in this disclosure, e.g., to achieve a more uniform distribution of target material across the sequencing surface in the flow cell.

[0012] Another example described herein is suitable for reducing or preventing template strand migration during on-flow cell amplification.

[0013] Thus, a seventh aspect disclosed herein is a method comprising the steps of: introducing sequenceable nucleic acid fragments into a flow cell, thereby seeding at least some of the sequenceable nucleic acid fragments to respective primers on a sequencing surface of the flow cell; removing unseeded sequenceable nucleic acid fragments from the flow cell; introducing an amplification mixture comprising a temperature-responsive material in liquid form into the flow cell; gelling the temperature-responsive material in liquid form; initiating amplification of the seeded sequenceable nucleic acid fragments to produce template strands, whereby the temperature-responsive material in gel form reduces diffusion of the template strands; liquefying the temperature-responsive material in gel form; and removing the temperature-responsive material in liquid form from the flow cell.

[0014] It should be understood that any features of the seventh aspect can be combined together in any desired manner. It should also be understood that any combination of features of the seventh aspect can be combined with any of the other aspects and / or embodiments disclosed herein to achieve the benefits described in this disclosure, including, for example, more even distribution of target material across the sequencing surface within the flow cell and reduced migration of template strands during flow cell amplification.

[0015] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numbers correspond to similar, but perhaps not identical, components. For purposes of brevity, reference numbers or features having a previously mentioned function may or may not be described in conjunction with other drawings in which they appear. [Brief explanation of the drawings]

[0016] [Figure 1A] 1 is a schematic diagram of an example target material disclosed herein. [Figure 1B]1 is a schematic diagram of an example target material disclosed herein. [Figure 1C] 1 is a schematic diagram of an example target material disclosed herein. [Figure 2A] FIG. 2 is a top view of an example of a flow cell. [Figure 2B] 2B is an enlarged cross-sectional view of an example of a flow channel and unpatterned sequencing surface taken along line 2B-2B of FIG. 2A. [Figure 2C] 2C is an enlarged cross-sectional view of an example of a flow channel and patterned sequencing surface taken along line 2C-2C of FIG. 2A. [Figure 2D] 2D is an enlarged cross-sectional view of another example of a flow channel and patterned sequencing surface taken along line 2D-2D of FIG. 2A. [Figure 3A] An example of the method disclosed herein is shown. [Figure 3B] An example of the method disclosed herein is shown. [Figure 4A] 10 illustrates another example of the method disclosed herein. [Figure 4B] 10 illustrates another example of the method disclosed herein. [Figure 5A] 10 illustrates yet another example of the method disclosed herein. [Figure 5B] 10 illustrates yet another example of the method disclosed herein. [Figure 6A] 10 illustrates yet another example of the method disclosed herein. [Figure 6B] 10 illustrates yet another example of the method disclosed herein. [Figure 7A] 10A-10C illustrate additional examples of the methods disclosed herein. [Figure 7B] 10A-10C illustrate additional examples of the methods disclosed herein. [Figure 8A] 10 illustrates yet another example of the method disclosed herein. [Figure 8B] 10 illustrates yet another example of the method disclosed herein. [Figure 9A] 1 shows an example of a method for reducing diffusion and convection of template strands during amplification. [Figure 9B] 1 shows an example of a method for reducing diffusion and convection of template strands during amplification. [Figure 9C] 1 shows an example of a method for reducing diffusion and convection of template strands during amplification. [Figure 10A] 1 shows the top sequencing surface of a flow cell containing a patterned sequencing surface. [Figure 10B] A bright field image of the complex immobilized on the bottom sequencing surface is shown. [Figure 11A] 1 is a histogram of molecular coverage of the upper and lower sequencing surfaces of one lane of a flow cell after sequencing has been performed. [Figure 11B] 1 is a graph showing the percentage of Q scores greater than Q30 (Y-axis) versus sequencing cycle number (X-axis) for the upper and lower sequencing surfaces of one lane after sequencing has been performed. [Figure 12A] 1 is a bar graph showing complex loading (beads / mm2, Y-axis) performed using two different loading solutions on the bottom of a flow cell treated with different concentrations (μM, X-axis) of alkyne biotin. [Figure 12B] 1 is a bar graph showing complex loading (beads / mm2, Y-axis) performed using two different loading solutions on top of a flow cell treated with different concentrations (μM, X-axis) of alkyne biotin. [Figure 13A] Graph showing target complex loading and actual complex loading (number of beads / mm 2 , Y-axis) at the bottom and top surfaces along the length (X-axis) of two different flow cell channels. [Figure 13B] Graph showing target and actual conjugate loading (number of beads / mm 2 , Y-axis) at the bottom and top surfaces along the length (X-axis) of two different flow cell channels. [Figure 14] Graph showing the target / expected conjugate loading, the actual conjugate loading (number of beads / mm, Y-axis) on the bottom and top surfaces along the length of one flow cell channel (X-axis), and a linear fit for each surface. DETAILED DESCRIPTION OF THE INVENTION

[0017] Some sequencing techniques utilize sequenceable nucleic acid fragments. In some instances, each sequenceable nucleic acid fragment comprises a portion of genetic material (fragment) and adapters at the 3' and 5' ends. The sequenceable nucleic acid fragments may be attached to a solid support to form a complex. In these instances, the use of a solid support may be desirable because it preserves adjacent information about the longer genetic material from which the fragment is generated. Other sequencing techniques utilize clustered solid supports, which contain clusters of templates attached to the solid support. In these instances, the use of a solid support may be desirable because amplification (template strand formation) can be performed outside the flow cell, and therefore the flow cell chemistry is simplified in that it does not include amplification primers. However, when these target materials (e.g., complexes or clustered solid supports) are used in a flow cell having two opposing sequencing surfaces (e.g., an upper surface / top face and a lower surface / bottom face), it has been found that the target materials tend to sink to the sequencing surface located at the bottom of the flow cell. Similar issues can arise with other target materials such as protein biomarkers, microbiota, lysates, etc. in flow cells with opposing surfaces.

[0018] Some examples of the methods disclosed herein provide a more balanced immobilization of target materials across two opposing sequencing surfaces. In some examples, the same type of target material is immobilized across two opposing sequencing surfaces. In other examples, two different target materials (having at least one different characteristic) are immobilized on each of the two opposing sequencing surfaces.

[0019] One example of a method disclosed herein utilizes a combination of fluids with different densities, where one fluid density allows target material (e.g., complexes, clustered solid supports) to migrate and be immobilized on one of the sequencing surfaces, and another fluid density allows target material to migrate and be immobilized on the other sequencing surface.

[0020] Another example of the method utilizes a combination of a fluid, a substantially uniform magnetic force, and a magnetically responsive target material (e.g., a solid support). In this example, the fluid is selected to have approximately the same density as the magnetically responsive target material. In this fluid, some of the target material sinks (and is immobilized on one of the sequencing surfaces), while other portions of the target material float. When a substantially uniform magnetic force is applied to the other sequencing surface, the floating target material migrates to the other sequencing surface and is immobilized there.

[0021] Yet another example of the methods disclosed herein utilizes two different target materials with different densities. Both target materials are contained in the same fluid. The density of one target material (with respect to the fluid) allows that target material (e.g., a complex, a clustered solid support) to migrate to and be immobilized on one of the sequencing surfaces, while the density of the other target material (with respect to the fluid) allows that target material to migrate to and be immobilized on the other sequencing surface.

[0022] Yet another example of the methods disclosed herein utilizes two different target materials having at least one different property, such as density, charge, magnetism, or a combination thereof, such that exposure to at least one condition causes the different target materials to migrate to respective ones of the opposing sequencing surfaces.

[0023] Immobilizing target material (eg, complexes, clustered solid supports, etc.) on both sequencing surfaces increases the overall utilization of the flow cell.

[0024] A more balanced distribution of immobilized target material across the two sequencing surfaces can improve downstream metrics obtained using the flow cell. In one example, a more balanced distribution of immobilized target material across the two sequencing surfaces can lead to improved sequencing metrics. In one example, the target material can include complexes. If the complexes are more evenly distributed across the two sequencing surfaces of the flow cell, library fragments released from the complexes will also seed more evenly across each sequencing surface. This seeding leads to the formation of individual clusters that are relatively localized with respect to the location of the complexes from which the clusters are formed. In another example, the target material can include clustered solid supports. If the clustered solid supports are more evenly distributed across the two sequencing surfaces of the flow cell, the clustered template strands will also be more evenly distributed. During sequencing, as nucleotides are incorporated into each template strand of the cluster, the individual clusters generate a "spatial cloud" of fluorescent signal. The even distribution can improve the readability of the spatial cloud.

[0025] Furthermore, loading both sequencing surfaces provides more area for generating their spatial clouds.

[0026] definition Terms used herein will be understood to take their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are set forth below.

[0027] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly dictates otherwise. As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude further, unrecited elements or method steps.

[0028] References throughout this specification to "one example," "another example," "an example," etc. mean that a particular element (e.g., a feature, structure, composition, configuration, and / or characteristic) described in connection with an example is included in at least one example described herein and may or may not be present in other examples. Furthermore, unless the context clearly dictates otherwise, it should be understood that the described elements with respect to any example may be combined in any suitable manner in the various examples.

[0029] As used throughout this disclosure, including the claims, the terms "substantially" and "about" are used to describe and account for small variations, such as those due to variations in processing, etc. The terms can refer to ±10% or less from the stated value, for example, ±5% or less from the stated value, ±2% or less from the stated value, ±1% or less from the stated value, ±0.5% or less from the stated value, ±0.2% or less from the stated value, ±0.1% or less from the stated value, or ±0.05% or less from the stated value.

[0030] Adapter: A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by ligation or tagmentation. Suitable adapter lengths can range from about 10 nucleotides to about 100 nucleotides, or from about 12 nucleotides to about 60 nucleotides, or from about 15 nucleotides to about 50 nucleotides. Adapters can comprise any combination of nucleotides and / or nucleic acids. In some examples, adapters can comprise a sequence complementary to at least a portion of a primer, e.g., a primer containing a universal nucleotide sequence (e.g., a P5 or P7 sequence). For example, an adapter at one end of a fragment comprises a sequence complementary to at least a portion of a first flow cell or solid support primer, and an adapter at the other end of the fragment comprises a sequence identical to at least a portion of a second flow cell or solid support primer. The complementary adapter can hybridize to the first flow cell or solid support primer, and the identical adapter is a template for its complementary copy that can hybridize to the second flow cell or solid support primer during clustering. In some examples, the adapter can comprise a sequencing primer sequence or a sequencing binding site. Different combinations of adaptors can be incorporated into a nucleic acid molecule, such as a DNA fragment.

[0031] Approximately equivalent: At least approximately equivalent means that the density of one component (e.g., a fluid) is less than 0.08 g / cm of the density of another component (e.g., a solid support). 3 In some cases, the densities of the two components are comparable.

[0032] Capture site or chemical capture site: A portion of a flow cell surface modified to possess chemical properties that allow for the localization of a target substance (e.g., a complex, a clustered solid support, a protein biomarker, etc.). In one example, a capture site can include a chemical capture agent (i.e., a material, molecule, or moiety that can attach, retain, or bind to a target molecule (e.g., a complex, a clustered solid support, a protein biomarker, etc.)). An example of a chemical capture agent is a member of a receptor-ligand binding pair (e.g., avidin, streptavidin, biotin, lectins, carbohydrates, nucleic acid-binding proteins, epitopes, antibodies, etc.) that can bind to a target material (or a linking moiety attached to the target material). Further examples of chemical capture agents include chemical reagents that can form electrostatic interactions, hydrogen bonds, or covalent bonds (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder reaction, etc.) with a target material.

[0033] Complex: A carrier, such as a solid support, and a sequenceable nucleic acid fragment attached to the carrier. The carrier can also include one member of a binding pair, the other member of which is part of the capture site.

[0034] Clustered solid support: A carrier, such as a solid support, to which multiple amplified template strands are attached. The multiple amplified template strands may be referred to as a "cluster."

[0035] Deposition: Any suitable application technique, which may be manual or automated, and which in some cases results in modification of surface properties. Generally, deposition may be carried out using evaporation techniques, coating techniques, grafting techniques, etc. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow-through coating, aerosol printing, screen printing, microcontact printing, inkjet printing, etc.

[0036] Depression: A discrete concave feature in a substrate or patterned resin having a surface opening at least partially surrounded by a gap area of ​​the substrate or patterned resin. Depressions can have a variety of shapes at the surface opening, such as, for example, a circle, an ellipse, a square, a polygon, a star (with any number of vertices), etc. The cross section of the depression taken perpendicular to the surface can be a curved shape, a square, a polygon, a hyperbola, a cone, an angled shape, etc. By way of example, a depression can be a well or two interconnected wells. A depression can also have a more complex structure, such as a ridge, a stepped structure, etc.

[0037] Each: When used in reference to a collection of items, each identifies an individual item in the collection, but does not necessarily refer to all of the items in the collection. Exceptions may occur where explicit disclosure or context clearly dictates otherwise.

[0038] External fixative: A gas, liquid, or viscous medium that is immiscible with the complexes introduced into the flow cell. A gaseous external fixative can be used to create liquid droplets around the complexes or samples. One 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 aspirate fluid from the flow cell, forming liquid droplets around the complexes immobilized within the flow cell. The droplets formed act as a diffusion barrier. A liquid or viscous medium is used to minimize the diffusion of the sequencing library released from the complexes. Because the sequencing library or other polynucleotides have little or no solvation in the external fixative, the external fixative can form a diffusion barrier. Examples of external fixatives in liquid form include hydrophobic oils such as mineral oil, silicone oil, perfluorinated oil, fluorocarbon oil (e.g., FC40), or combinations thereof. Examples of external fixatives in viscous medium form include buffers containing polymers (e.g., polyethylene glycol, polyvinylpyrrolidone, etc.), dextran, sucrose, glycerol, etc. In some instances, the viscous medium is a temperature-responsive gel. The temperature-responsive gel is non-viscous at the non-seeding temperature and transforms into a viscous medium at the seeding temperature. Examples of temperature-responsive gels include poly(N-isopropylacrylamide) and polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) / Laponite nanoparticle composites.

[0039] Flow cell: A container having a chamber (e.g., a flow channel) in which a reaction can occur, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some instances, the chamber allows for detection of the reaction occurring within the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of arrays, optically labeled molecules, etc.

[0040] Flow channel: A region defined between two bonded or otherwise attached components that can selectively receive a liquid sample. In some examples, a flow channel may be defined between two patterned or unpatterned sequencing surfaces and thus may be in fluid communication with one or more components of the sequencing surfaces.

[0041] Fragment: A portion or segment of genetic material (e.g., DNA, RNA, etc.). Contiguous library fragments are small segments of a fragmented longer nucleic acid sample in which contiguous information in the longer nucleic acid sample is stored.

[0042] Nucleic Acid Molecule or Sample: A polymeric form of nucleotides of any length, which may contain ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term may refer to single- or double-stranded polynucleotides.

[0043] A "template" nucleic acid molecule (or strand) can refer to the sequence to be analyzed. Clusters of template strands comprise the amplicons of library fragments.

[0044] Nucleotides in nucleic acid molecules can include naturally occurring nucleic acids and their functional analogs. Examples of functional analogs can hybridize to nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone linkages, including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). Analog structures can have alternative sugar moieties, including any of a variety known in the art. Nucleotides can contain natural or unnatural bases. Natural DNA can contain one or more of adenine, thymine, cytosine, and / or guanine, while natural RNA can contain one or more of adenine, uracil, cytosine, and / or guanine. Any unnatural base can be used, such as locked nucleic acids (LNA) and bridged nucleic acids (BNA).

[0045] Primer: A nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a library fragment. In one example, an amplification primer can serve as the starting point for template amplification and cluster generation. In another example, a synthesized nucleic acid (template) strand can contain a site to which a primer (e.g., a sequencing primer) can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid strand. Any primer can contain any combination of nucleotides or their analogs. In some examples, a primer is a single-stranded oligonucleotide or polynucleotide. The length of a primer can be any number of bases and can contain various non-natural nucleotides. In one example, a sequencing primer is a short strand ranging from 10 to 60 bases or 20 to 40 bases.

[0046] Sequenable nucleic acid fragment: A portion of genetic material with adapters at the 3' and 5' ends. In a sequenceable nucleic acid fragment, each adapter contains a known universal sequence (e.g., complementary to or identical to at least a portion of a primer on a flow cell) and a sequencing primer sequence. Both adapters can also contain index (barcode or tag) sequences. In one example, one end (e.g., containing the P5' or P5 sequence) can contain a bead index, and the other end (containing the P7 or P7' sequence) can contain a sample index. Sequenable nucleic acid fragments can be attached to a solid support via transposon insertion, where the inserted DNA molecule is immobilized on the surface of the solid support (e.g., a bead), or directly immobilized via a binding pair or other cleavable linker, or attached by hybridization, with the complementary adapter sequence present on the surface of the solid support.

[0047] Sequencing surface: The surface of a flow cell on which sequencing can be performed. In some examples, the sequencing surface comprises a polymeric hydrogel having one or more types of amplification primers grafted thereto. In these examples, the sequencing surface may also comprise capture sites for immobilizing complexes at or near the amplification primers. In other examples, the sequencing surface comprises capture sites for immobilizing clustered solid supports.

[0048] Solid support: A small body made of a rigid or semi-rigid material, having a shape characterized by, for example, a sphere, an oval, a microsphere, or other recognizable particulate shape, whether having regular or irregular dimensions. In some instances, the solid support may have a sequencing library attached thereto. In other instances, the solid support may have clusters of template strands attached thereto.

[0049] Target material: The substance that is immobilized on the flow cell surface.

[0050] Transposome: A complex formed between an integration enzyme (e.g., an integrase or transposase) and a nucleic acid containing an integration recognition site (e.g., a transposase recognition site).

[0051] In the examples disclosed herein, the target material is introduced into a flow cell that includes two opposing sequencing surfaces. The target material and the flow cell are now described, followed by various exemplary methods for immobilizing the target material on each of the two opposing sequencing surfaces.

[0052] target material Examples of target materials 11 are shown in Figures 1A-1C. In the embodiments disclosed herein, any target material 11 that can be immobilized on the surface of a flow cell can be utilized. By way of example, target material 11 can be complexes 10A, 10B defined herein (see Figures 1A and 1B), clustered solid supports 13 defined herein (see Figure 1C), other DNA libraries from a particular sample, cells, oligonucleotide-conjugated proteins bound to a solid support, protein biomarkers, microbiomes, etc. The following description provides some examples of complexes 10A, 10B, and clustered solid supports 13.

[0053] Complex Some exemplary complexes 10A and 10B are shown in Figures 1A and 1B, respectively. In examples of the methods disclosed herein, complexes 10A, 10B include solid supports 12, 12' and sequenceable nucleic acid fragments 14, 14', 14" attached to solid supports 12, 12'.

[0054] In exemplary methods utilizing fluids with different densities, or target materials 11 with different densities, or a combination of uncharged target materials 11, the solid support 12 can be, but is not limited to, hydrogel; glass (e.g., controlled pore glass beads); plastic, such as acrylic, polystyrene, or a copolymer of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (TEFLON® from The Chemours Co.); polysaccharides or cross-linked polysaccharides, such as agarose, SEPHAROSE® beads (a cross-linked beaded form of agarose available from Cytivia), or SEPHADEX® beads (a cross-linked beaded form of dextran available from Cytivia); nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metal; inorganic glass; fiber optic bundles; or various other polymers. Some examples of the solid support 12 can have the form of solid beads, porous beads, or hollow beads.

[0055] In an example of a method utilizing a combination of fluid and magnetic forces, the solid support 12' is a magnetically responsive material. A "magnetically responsive" material responds to a magnetic field. Examples of magnetically responsive solid supports include or are composed of a magnetically responsive material. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as Fe3O4, BaFe 12 O 19, CoO, NiO, Mn2O3, Cr2O3, and metal oxides such as CoMnP. One commercially available example includes ThermoFisher Scientific's DYNABEAD™ M-280 Streptavidin (superparamagnetic beads coated with streptavidin). In some examples, the magnetically responsive material is embedded in the shell of a polymer bead. In other examples, the magnetically responsive material is in bead form and coated with a passivating material such as silicon oxide or silicon nitride. In an exemplary method utilizing two different target materials 11, one of the target materials 11 can include any of the magnetically responsive solid supports 12′ disclosed herein.

[0056] In examples of methods utilizing an electric field for immobilization, the solid support 12 of the target material 11 can be positively or negatively charged. In these examples, any of the described solid supports 12 can be used and can be coated or functionalized to impart the desired charge. Either small molecules or polymers can be used to impart a charge to the solid support 12. For example, any of the solid supports 12 (e.g., polystyrene, silica, etc.) can be functionalized with amines to impart a positive charge. Any primary, secondary, or tertiary amine can be used. Examples of suitable amines include aminosilane, polylysine, or chitosan. As another example, any of the solid supports 12 (e.g., polystyrene, silica, SEPHADEX®, etc.) can be functionalized with carboxyl or sulfate groups to impart a negative charge to the support. As yet another example, any of the solid supports 12 (e.g., polystyrene, silica, SEPHADEX®, etc.) can be coated with polyglutamic acid to impart a negative charge to the support.

[0057] Although not shown in Figures 1A and 1B, solid supports 12, 12' may be functionalized with one member of a binding pair. A "binding pair" refers to two agents (e.g., materials, molecules, moieties) that can attach to one another. In this example, a member on solid supports 12, 12' is a binding pair with another member located on the sequencing surface of a flow cell. In other examples, solid supports 12, 12' may be chemically bonded to the sequencing surface of a flow cell.

[0058] Functionalization of the solid support 12, 12' can involve coating the solid support 12, 12' with a binding pair member or forming a bond between a binding pair member and a functional group on the surface of the solid support 12, 12'. Exemplary binding pair members include members of receptor-ligand binding pairs (e.g., avidin, streptavidin, biotin, lectins, carbohydrates, nucleic acid-binding proteins, epitopes, antibodies, etc.) that can bind to other binding pair members located on the sequencing surface of the flow cell. Binding pair members can also be chemical reagents that can form electrostatic interactions, hydrogen bonds, or covalent bonds (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder reaction, etc.). Any form of chemical bond can also attach the solid support 12, 12' to the sequencing surface of the flow cell. Often, a reversible or cleavable interaction is desired, so the solid support 12, 12' can be removed prior to sequencing.

[0059] In the example complexes 10A, 10B, sequenceable nucleic acid fragments 14, 14', 14" are attached to solid supports 12, 12'. Each sequenceable nucleic acid fragment 14, 14', 14" comprises a portion of longer genetic material (e.g., fragment 16, 16', 16") with adapters (e.g., 18, 18', 18", 22, 22', 22") at the 3' and 5' ends. The sequenceable fragments 14, 14', 14" can be prepared using any library preparation technique that fragments longer genetic material and incorporates desired adapters 18, 18', 18", 22, 22', 22" at the ends of the fragments 16, 16', 16". Some suitable library preparation techniques are described with reference to Figures 1A and 1B. However, it should be understood that other library preparation techniques can also be used.

[0060] 1A shows an example of a complex 10A comprising sequenceable nucleic acid fragments 14, 14', which comprise fragments 16, 16' from a larger nucleic acid sample, the contiguity of which is preserved on solid supports 12, 12'. While an exemplary method for making complex 10A is described herein, it should be understood that other methods can be used, provided that the sequenceable nucleic acid fragments 14, 14' are attached to solid supports 12, 12'.

[0061] In one example method for forming the complex 10A shown in FIG. 1A, an adapter sequence 18, 18′ is attached to the solid support 12, 12′ via one member 20 of a binding pair. In one example, the adapter sequence 18, 18′ can include a first sequence (P5′) that is complementary to at least a portion of a first sequencing primer sequence (e.g., a read 1 sequencing primer sequence) and one of the amplification primers (e.g., P5) on the flow cell (shown in FIGS. 2A, 2B, and 2C). The adapter sequence 18, 18′ can also include an index sequence or barcode sequence. The adapter sequence 18, 18′ can be attached to one member 20 of a binding pair (e.g., biotin), which in turn can be attached to the surface of the solid support 12, 12′ that includes the other member of the binding pair (e.g., avidin, streptavidin, etc.). In this example, the members of the binding pair on the solid support 12, 12' are i) bound to the member 20 used to bind the sequenceable nucleic acid fragments 14, 14' and ii) bound to the sequencing surface of a flow cell. In other examples, the solid support 12, 12' can be functionalized with two different binding pair members, for example, i) one of which can bind to the member 20 used to attach the sequenceable nucleic acid fragments 14, 14' and ii) the other of which can bind to the sequencing surface of a flow cell.

[0062] In this example, transposome complexes (not shown) can also be attached to the solid supports 12, 12′ at the beginning of the library preparation method. Before loading the transposome complexes onto the solid supports 12, 12′, partial Y adapters can be mixed with a transposase enzyme (e.g., two Tn5 molecules) to form transposase complexes. The partial Y adapters can include two mosaic end sequences that hybridize to each other. One of the mosaic end sequences is referred to as a free mosaic end sequence because it has two free ends; for example, one end can be attached to adapter 18, 18′, and the other end can be attached to fragmented DNA strands 16, 16′ during tagging. One end of the mosaic end sequence can be attached to another adapter (e.g., 22, 22′), which includes a second sequence (P7) identical to a second sequencing primer sequence (e.g., read 2 sequencing primer sequence) and at least a portion of another amplification primer (P7) on the flow cell. During amplification, the identical sequence allows the formation of a copy complementary to at least a portion of the other amplification primer (P7) on the flow cell. The adapter sequences 22, 22' are not attached to the fragmented DNA strands 16, 16' during tagging.

[0063] Loading the transposome complexes onto the solid support 12, 12' can include mixing the transposome complexes with the solid support 12, 12' and exposing the mixture to appropriate conditions for ligating one of the free ends of the free mosaic ends to the 3' end of the adapter sequence 18, 18'. Individual transposome complexes can be attached to each of the adapter sequences 18, 18' on the solid support 12, 12'.

[0064] Next, in this exemplary method of forming complex 10A, a tagging process can be performed. A fluid (e.g., tagging buffer) containing a longer nucleic acid sample (e.g., DNA) can be added to solid supports 12, 12′ with adapter sequences 18, 18′ and transposome complexes bound thereto. When the sample contacts the transposome complexes, the longer nucleic acid sample is tagged. The longer nucleic acid sample is fragmented into fragments 16, 16′, each tagged at its 5′ end with a partial Y adapter (e.g., by ligation of the other free end of the free mosaic end sequence). Sequential tagging of the longer nucleic acid sample results in multiple bridge structure molecules between transposome complexes. The bridge structure molecules wrap around solid supports 12, 12′. The transposome complexes maintain the proximity of the longer nucleic acid sample as bridge structure molecules.

[0065] The transposase enzyme can then be removed by sodium dodecyl sulfate (SDS) treatment or heat or proteinase K digestion. Removal of the transposase enzyme leaves the contiguity-preserved fragments 16, 16' attached to the solid support 12, 12'.

[0066] To complete the sequenceable fragments 14, 14', further extension and ligation is performed to ensure that the sample fragments 16, 16' are attached to sequences 22 and 22'. The resulting complex 10A is shown in Figure 1A.

[0067] Each sequenceable nucleic acid fragment 14, 14' comprises a contiguous library fragment 16, 16' attached at both ends to respective adapter sequences 18 and 22 or 18' and 22'. The adapter sequence 18, 18' is initially attached to the solid support 12, 12' and comprises a first sequencing primer sequence and a first sequence complementary to one of the flow cell primers. The adapter sequence 18, 18' is attached to one member 20 of a binding pair. The adapter sequence 22, 22' is from a partial Y adapter and comprises a second sequence identical to another flow cell primer and a second sequencing primer sequence. Because each sequenceable nucleic acid fragment 14, 14' contains an adapter suitable for amplification (e.g., bridge amplification) and sequencing, PCR amplification is not performed. These fragments 14, 14' are therefore sequenceable. Furthermore, because the contiguous library fragments 16, 16' are derived from the same long nucleic acid sample, the contiguous nature of the original sample is preserved, making the library fragments 14, 14' suitable for concatenated long read applications.

[0068] FIG. 1B shows another complex 10B including a solid support 12, 12′ and a sequenceable nucleic acid fragment 14″ attached to the solid support 12, 12′. In one example, a PCR-free nucleotide library is generated in a tube, and the library is then hybridized to the solid support 12, 12′ in the tube. In the example shown in FIG. 1B, adapters 18″, 22″ are added to the library fragment 16″ in the tube, a primer having one member of a binding pair 20 is hybridized to the adapter 18″ in the tube, and the sequenceable nucleic acid fragment 14″ is then attached to the solid support 12, 12′ via the member of the binding pair 20. In another example, the solid support 12, 12′ can have a primer attached to the support via a binding pair (e.g., avidin on the support 12, 12′ and biotin attached to the primer). These primers hybridize to adaptors 18" attached to the library fragments 16" (thus the primer and binding pair member are at one end of the fragment but not the other). In yet another example, extension can be performed using a strand-displacing enzyme, resulting in a fully double-stranded library (e.g., no forks or Y adaptors, as shown in Figure 1B).

[0069] As mentioned above, other library preparation techniques can also be used. For example, ligation-based library preparation techniques can be used where complementary adapter sequences are immobilized on a flow cell. As another example, mRNA can be immobilized to a solid support 12, 12' via polyA tail hybridization.

[0070] Clustered solid supports An example of a clustered solid support 13 is shown in Figure 1C. The clustered solid support 13 includes solid supports 12, 12' and template strands 64 attached to the solid supports 12, 12' via primers 42 or 42'.

[0071] Any example of a solid support 12, 12' can be used as the core of the clustered solid support 13. The type of solid support 12, 12' and its property / properties (e.g., density, charge, magnetism, etc.) can vary depending on the immobilization method used.

[0072] Although not shown in Figure 1C, similar to the conjugates 10A and 10B shown in Figures 1A and 1B, the solid supports 12, 12' may be functionalized with one member of a binding pair for attachment to a capture site on a flow cell.

[0073] As shown in FIG. 1C, this example of a solid support 12, 12' is functionalized with a primer 42, 42'. The primer 42, 42' may be an amplification primer 42, 42' that can be immobilized to the solid support 12, 12' by a single-point covalent bond or a strong non-covalent interaction at or near the 5' end of the primer 42, 42'. This attachment allows i) the adapter-specific portion of the primer 42, 42' to freely anneal to its cognate sequenceable nucleic acid fragment, and ii) the 3' hydroxyl group is free for primer extension. At or near the 5' end, the primer 42, 42' contains a chemically modifiable functional group capable of covalent bonding or a strong non-covalent interaction. Examples of chemically modifiable functional groups include thiol, azide, alkyne, amino, biotin, etc.

[0074] Specific examples of suitable primers 42, 42' include the P5 and P7 primers used on the surface of commercially available flow cells sold by Illumina Inc. for sequencing on HiSeq™, HiSeqX™, MiSeq™, MiSeqDX™, MiNISeq™, NextSeq™, NextSeqDX™, NovaSeq™, Genome Analyzer™, ISEQ™, and other instrument platforms. Either of the P5 and P7 primers may be grafted onto the solid supports 12, 12', respectively.

[0075] In one example, grafting the primer 42, 42' to the solid support 12, 12' may include dunk coating, which may involve immersing the solid support 12, 12' in a primer solution or mixture, which may include the primer 42, 42', water, a buffer, and a catalyst. Other grafting techniques may include spray coating, droplet dispensing, or another suitable method of attaching the primer 42, 42' to the solid support 12, 12'. Using any of the grafting methods, the primer 42, 42' reacts with a reactive group on the solid support 12, 12'.

[0076] During grafting, the chemically modifiable functional groups of the primers 42, 42′ react or interact with reactive groups on the solid support 12, 12′. The following are examples of reactions or interactions that can occur during grafting: reacting an azide (e.g., succinimidyl (NHS) ester)-terminated primer with hydrazine on the surface of the solid support 12, 12′, or reacting an alkyne-terminated primer with an azide on the surface of the solid support 12, 12′, or reacting an amino-terminated primer with an activated carboxylate group or NHS ester on the surface of the solid support 12, 12′, or reacting a thiol-terminated primer with an alkylating reactant (e.g., iodoacetamine or maleimide) on the surface of the solid support 12, 12′, or reacting a phosphoramidite-terminated primer with a thioether on the surface of the solid support 12, 12′, or interacting a biotin-modified primer with streptavidin on the surface of the solid support 12, 12′. Some nucleic acid primers 42, 42' can be captured on silica beads in the presence of chaotropic agents (KI, NI, or NaSCN). As a specific example, primers terminated with dibenzocyclooctyne (alkyne-containing DBCO) can be used for copper-free click grafting.

[0077] To generate template strand 64 on solid support 12, 12′, library templates can first be prepared from any nucleic acid sample (e.g., a DNA sample or an RNA sample). When an RNA sample is used, it is first converted to a complementary deoxyribonucleic acid (cDNA) sample. This conversion can be performed using reverse transcription, which utilizes a reverse transcriptase enzyme. In some examples, a kit for reverse transcription and double-strand synthesis is used. In these examples, a high-capacity cDNA reverse transcription kit from ThermoFisherScientific can be used. In other examples, a kit for reverse transcription and template switching (for the second strand) is used. In these examples, a template-switching RT enzyme mix from New England Biolabs can be used.

[0078] The DNA or cDNA sample can then be fragmented into single-stranded, similarly sized (e.g., less than 1000 bp) fragments. During preparation, adapters can be added to the ends of these fragments. By reducing cycle amplification, different motifs can be introduced into the adapters, such as sequencing binding sites, indexes, and regions complementary to or identical to the primers 42, 42' on the solid supports 12, 12'. The final library template includes DNA or cDNA fragments and adapters on both ends. In some examples, fragments from a single nucleic acid sample have the same adapters added to the fragments.

[0079] Multiple library templates can be introduced onto multiple solid supports 12, 12′. The library templates hybridize to one of two types of primers 42, 42′ immobilized on each solid support 12, 12′. Cluster generation can then be performed. In one example of cluster generation, the library templates on the solid supports 12, 12′ are copied from the hybridized primers by 3′ extension using a high-fidelity DNA polymerase. The original library templates are denatured, and copies (e.g., template strands 64) remain immobilized on the solid supports 12, 12′, e.g., via primer 42, as shown in FIG. 1C. Isothermal bridge amplification or some other form of amplification can be used to amplify the immobilized copies. For example, the copied templates loop over and hybridize to adjacent complementary primers (e.g., primer 42′), and a polymerase copies the copied templates to form a double-stranded bridge structure, which is denatured to form two single strands. These two strands loop over and hybridize to adjacent complementary primers 42, 42', where they are extended again, forming two new double-stranded loops. This process is repeated for each template copy through cycles of isothermal denaturation and amplification, creating dense clonal clusters. Each cluster of double-stranded bridge structures is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. Clustering results in the formation of several template polynucleotide strands 64 on the solid support 12, 12'. An example of this clustering is bridge amplification, which is one example of a possible amplification.

[0080] Flow cell A top view of an example flow cell 24 is shown in FIG. 2A. As described herein, flow cell 24 includes two opposing sequencing surfaces. Examples of unpatterned sequencing surfaces 30, 30′ are shown in FIG. 2B, examples of patterned sequencing surfaces 32, 32′ are shown in FIG. 2C, and another example of patterned sequencing surfaces 31, 31′ is shown in FIG. 2D. Unpatterned sequencing surfaces 30, 30′ and patterned sequencing surfaces 32, 32′ include primers 42, 42′ and thus can be utilized with target material 11 to introduce library fragments to be amplified in flow cell 24. Other sequencing surfaces, such as patterned sequencing surfaces 31, 31′, do not include primers 42, 42′ and therefore can be utilized with clustered solid supports 13.

[0081] Each sequencing surface 30, 30' or 32, 32' or 31, 31' is supported by a substrate (generally shown as 26 in FIG. 2A), and a flow channel (generally shown as 28 in FIG. 2A) is defined between sequencing surfaces 30, 30' or 32, 32' or 31, 31'.

[0082] The substrate 26 can be a single layer / material. Examples of single layer substrates are shown in FIG. 2B with reference numerals 26A and 26A'. Examples of suitable single layer substrates 26A, 26A' include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefin / cycloolefin polymers (COP) (such as ZEONOR® from Zeon), polyamides, etc.), nylon (polyamide), ceramic / ceramic oxide, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (Ta2O5) or other tantalum oxides (TaO x), hafnium oxide (HfO2), carbon, metals, inorganic glasses, etc.

[0083] The substrate 26 may also be a multilayer structure. Examples of multilayer substrates are shown in Figures 2C and 2D as reference numerals 26B and 26B'. Some examples of multilayer structures 26B, 26B' ​​include glass or silicon with a coating layer of tantalum oxide or another ceramic oxide on the surface. With particular reference to Figures 2C and 2D, another example of a multilayer structure 26B, 26B' ​​includes a lower support 34, 34' having a patterned resin 36, 36' thereon. Yet another example of a multilayer substrate 26B, 26B' ​​may include a silicon-on-insulator (SOI) substrate.

[0084] In one example, the substrate 26 (whether single layer or multilayer) may have a rectangular sheet or panel having a diameter ranging from about 2 mm to about 300 mm, or a maximum dimension up to about 10 feet (~3 meters). In one example, the substrate 26 is a wafer having a diameter ranging from about 200 mm to about 300 mm. In another example, the substrate 26 is a die having a width ranging from about 0.1 mm to about 10 mm. While exemplary dimensions are provided, it should be understood that a substrate 26 having any suitable dimensions can be used. As another example, a panel can be used that is a rectangular support having a surface area greater than a 300 mm round wafer.

[0085] In the example shown in FIG. 2A , the flow cell 24 includes flow channels 28. While several flow channels 28 are shown, it should be understood that any number of channels 28 can be included in the flow cell 24 (e.g., a single channel 28, four channels 28, etc.). In the examples disclosed herein, each flow channel 28 is a region defined between two sequencing surfaces (e.g., 30 and 30′, or 32 and 32′, or 31 and 31′) and two attached substrates (e.g., 26A and 26A′, or 26B and 26B′). Fluids described herein can be introduced into and removed from the flow channels 28 via inlets and outlets, respectively. Each flow channel 28 can be isolated from each other within the flow cell 24, such that fluids introduced into any particular flow channel 28 do not flow into adjacent flow channels 28.

[0086] A portion of the flow channel 28 may be defined in the substrate 26 using any suitable technique, depending in part on the material of the substrate 26. In one example, a portion of the flow channel 28 is etched into the glass substrate 26. In another example, a portion of the flow channel 28 may be patterned into the resin 36, 36' of the multilayer substrate 26B, 26B' ​​using photolithography, nanoimprint lithography, or the like. In yet another example, a separate material (e.g., material 50 in FIGS. 2B, 2C, and 2D) may be applied to the substrate 26, the separate material defining at least a portion of the walls of the flow channel 28.

[0087] In one example, the flow channels 28 have a rectangular configuration. The length and width of the flow channels 28 may be smaller than the length and width, respectively, of the substrate 26, such that a portion of the substrate surface surrounding the flow channels 28 is available for attachment to another substrate 26. In some cases, the width of each flow channel 28 may be at least about 1 mm, at least about 2.5 mm, at least about 5 mm, at least about 7 mm, at least about 10 mm, or more. In some cases, the length of each flow channel 28 may be at least about 10 mm, at least about 25 mm, at least about 50 mm, at least about 100 mm, or more. The width and / or length of each flow channel 28 may be greater than, less than, or between the values ​​specified above. In another example, the flow channels 28 are square (e.g., 10 mm x 10 mm).

[0088] The depth of each flow channel 28 can be as small as several monolayers thick, for example, when using microcontact, aerosol, or inkjet printing to deposit a separate material (e.g., material 50) defining the channel walls. In other examples, the depth of each flow channel 28 can be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. In one example, the depth can range from about 10 μm to about 100 μm. In another example, the depth is about 5 μm or less. It should be understood that the depth of each flow channel 28 can be greater than, less than, or between the values ​​specified above. The depth of the flow channel 28 can also vary along the length and width of the flow cell 24, for example, when patterned sequencing surfaces 32, 32′ or 31, 31′ are used.

[0089] 2B shows a cross-sectional view of flow cell 24 including opposing unpatterned sequencing surfaces 30, 30′. In one example, each of these surfaces 30, 30′ can be prepared on substrates 26A, 26A′, which can then be attached to one another to form the example flow cell 24. Any suitable bonding material 50, such as an adhesive, a radiation-absorbing material that aids bonding, or the like, can be used to bond substrates 26A, 26B together.

[0090] 2B, a portion of flow channel 28 is defined in each of single-layer substrates 26A, 26A'. For example, each substrate 26A, 26A' may have a recessed region 38, 38' defined therein into which components of sequencing surface 30, 30' may be incorporated. It should be understood that any space within recessed region 38, 38' that is not occupied by components of sequencing surface 30, 30' may be considered to be part of flow channel 28.

[0091] The sequencing surface 30, 30' comprises a polymer hydrogel 40, 40', amplification primers 42, 42' attached to the polymer hydrogel 40, 40', and chemical capture sites 44, 44'.

[0092] Examples of polymeric hydrogels 40, 40′ include acrylamide copolymers such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide), PAZAM. PAZAM and some other forms of acrylamide copolymers are represented by the following structure (I): [ka] (In the formula, R A is selected from the group consisting of azide, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol; R B is H or optionally substituted alkyl; R C , R D , and R E are each independently selected from the group consisting of H and optionally substituted alkyl; -(CH2) p - may be optionally replaced by p is an integer ranging from 1 to 50; n is an integer ranging from 1 to 50,000, and m is an integer ranging from 1 to 100,000).

[0093] Those skilled in the art will recognize that the arrangement of the repeating features "n" and "m" in structure (I) is representative and that the monomer subunits may be present in any order in the polymer structure (e.g., random, block, patterned, or combinations thereof).

[0094] The molecular weight of PAZAM and other forms of acrylamide copolymers can range from about 5 kDa to about 1500 kDa, or from about 10 kDa to about 1000 kDa, or in a particular example, can be about 312 kDa.

[0095] In some instances, PAZAM and other forms of acrylamide copolymers are linear polymers. In other instances, PAZAM and other forms of acrylamide copolymers are lightly crosslinked polymers.

[0096] In other examples, the polymer hydrogel 40, 40' can be a variation of structure (I). In one example, the acrylamide units are N,N-dimethylacrylamide. [ka] In this example, the acrylamide unit of structure (I) can be replaced by [ka] where R D , R E , and R F are each H or C1-C6 alkyl, and R G and R Hare each C1-C6 alkyl (rather than H as in acrylamide). In this example, q can be an integer ranging from 1 to 100,000. In another example, in addition to the acrylamide units, N,N-dimethylacrylamide can be used. In this example, structure (I) contains, in addition to the repeating features "n" and "m", [ka] where R D , R E , and R F are each H or C1-C6 alkyl, and R G and R H is a C1-C6 alkyl. In this example, q can be an integer ranging from 1 to 100,000.

[0097] As another example of the polymeric hydrogel 40, 40′, the repeating feature “n” in structure (I) may be replaced with a heterocyclic azide group-containing monomer having structure (II): [ka] In the formula, R 1 is H or C1-C6 alkyl, and R 2 is H or C1-C6 alkyl; L is a linker comprising a linear chain of 2-20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, with 10 optional substituents on the carbons and an optional nitrogen atom in the chain; E is a linear chain of 1-4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, with optional substituents on the carbons and any nitrogen atoms in the linear chain; A is an N-substituted amide with H or C1-C4 alkyl attached to the N; and Z is a nitrogen-containing heterocycle. Examples of Z include 5-10 membered rings that exist as single ring structures or fused structures. Some specific examples of Z include pyrrolidinyl, pyridinyl, or pyrimidinyl.

[0098] As yet another example, polymer hydrogels 40, 40′ may include repeat units of structures (III) and (IV), respectively: [ka] where R 1a , R 2a , R 1b and R 2b each independently selected from hydrogen, optionally substituted alkyl, or optionally substituted phenyl; R 3a and R 3b each is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7-C14 aralkyl; 1 and L 2 are independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0099] It should be understood that other molecules can also be used to form the polymeric hydrogels 40, 40′, provided they are functionalized to graft oligonucleotide primers 42, 42′. Other examples of suitable polymer layers include colloidal structures such as agarose, polymer mesh structures such as gelatin, or cross-linked polymer structures such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA), or azide-decomposed versions 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 suitable polymeric hydrogels 42 include mixed copolymers of acrylamide and acrylate. Various polymeric structures containing acrylic monomers (e.g., acrylamide, acrylate, etc.) can be utilized in the embodiments disclosed herein, including star polymers, star or star-block polymers, branched polymers including dendrimers, etc. For example, monomers (e.g., acrylamide, etc.) can be incorporated into the branches (arms) of a star polymer, either randomly or in blocks.

[0100] To introduce the polymer hydrogel 40, 40′ into the recessed regions 38, 38′, a mixture of the polymer hydrogel 40, 40′ can be created and then applied to the respective substrates 26A, 26A′ (which define the recessed regions 38, 38′). In one example, the polymer hydrogel 40, 40′ can be present in a mixture (e.g., with water, or a mixture of ethanol and water). The mixture can then be applied to the respective substrate surfaces (including the recessed regions 38, 38′) using spin coating, dipping or dip coating, flow of material under positive or negative pressure, or another suitable technique. These types of techniques result in blanket deposition of the polymer hydrogel 40, 40′ onto the respective substrates 26A, 26A′ (e.g., into the recessed regions 38, 38′ and adjacent interstitial regions 46, 46′). Other selective deposition techniques (including, for example, masks, controlled printing techniques, etc.) can be used to specifically deposit polymeric hydrogel in the recessed regions 38, 38' but not in the interstitial regions 46, 46'.

[0101] In some examples, the substrate surface (including recessed regions 38, 38') can be activated and then the mixture (including polymer hydrogel 40, 40') can be applied thereto. In one example, a silane or silane derivative (e.g., norbornene silane) can be deposited on the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface can be exposed to plasma ashing, thereby generating surface-activating agents (e.g., -OH groups) that can attach to the polymer hydrogel 40, 40'.

[0102] Depending on the chemical nature of the polymer hydrogel 40, 40', the applied mixture may be subjected to a curing process. In one example, curing may occur at temperatures ranging from room temperature (e.g., about 25°C) to about 95°C for times ranging from about 1 millisecond to about several days.

[0103] Polishing may be performed to remove the polymer hydrogel 40, 40' from the gap regions 46, 46' surrounding the recessed regions 38, 38' and leave the polymer hydrogel 40, 40' at least substantially intact on the surfaces of the recessed regions 38, 38'.

[0104] The sequencing surface 30, 30' also includes amplification primers 42, 42' attached to a polymeric hydrogel 40, 40'.

[0105] A grafting process can be performed to graft the amplification primers 42, 42′ to the polymer hydrogel 40, 40′ in the recessed regions 38, 38′. In one example, the amplification primers 42, 42′ can be immobilized to the polymer hydrogel 40, 40′ by a single-point covalent bond or strong non-covalent interaction at or near the 5′ end of the primer 42, 42′. This attachment allows i) the adapter-specific portion of the primer 42, 42′ to be free to anneal to its cognate sequenceable nucleic acid fragment, and ii) the 3′ hydroxyl group is free for primer extension. Any suitable covalent bond or strong non-covalent interaction can be used for this purpose. Examples of terminal primers that can be used include alkyne-terminated primers (e.g., which can be attached to the azide surface moiety of the polymer hydrogel 40, 40′), or azide-terminated primers (e.g., which can be attached to the alkyne surface moiety of the polymer hydrogel 40, 40′), or any of the other terminal primers described with respect to the clustered solid support 13.

[0106] Specific examples of suitable primers 42, 42' include P5 and P7 primers, either of which may be grafted onto the polymeric hydrogels 40, 40', respectively.

[0107] In one example, grafting can be by flow-through deposition (e.g., using a temporarily bonded lid), dunk coating, spray coating, droplet dispensing, or another suitable method of attaching the primer 42, 42′ to the polymer hydrogel 40, 40′. Each of these exemplary techniques can utilize a primer solution or mixture that can include the primer 42, 42′, water, a buffer, and a catalyst. Using any of the grafting methods, the primer 42, 42′ reacts with reactive groups on the polymer hydrogel 40, 40′ in the recessed regions 38, 38′ and has no affinity for the surrounding substrate 26A, 26A′. Thus, the primer 42, 42′ selectively grafts to the polymer hydrogel 40, 40′.

[0108] In the example shown in Figure 2B, the chemical capture sites 44, 44' comprise a chemical capture agent attached or applied to at least a portion of the polymer hydrogel 40, 40'. Any example of a chemical capture agent disclosed herein can be used. For example, the chemical capture agent can be a member of a binding pair, where the other member of the binding pair is attached to the solid support 12, 12'.

[0109] In some examples, free functional groups on the polymer hydrogel 40, 40′ (e.g., those not attached to primers 42, 42′) can be functionalized with a chemical capture agent such that several chemical capture sites 44, 44′ are formed across the surface of the polymer hydrogel 40, 40′. In one example, click chemistry can be used to covalently attach an alkyne-PEG-biotin linker or alkyne-biotin free azide group to the free azide on the polymer hydrogel 40, 40′. In another example, primers complementary to the amplification primers 42, 42′ can have a chemical capture agent attached to them. These complementary primers can be hybridized to several amplification primers 42, 42′ to form chemical capture sites 44, 44′.

[0110] In another example, the chemical scavenger can be deposited at the desired location using microcontact printing, aerosol printing, etc. to form the chemical scavenging sites 44, 44'. In yet another example, a mask (e.g., photoresist) can be used to define the spaces / locations where the chemical scavenger can be deposited, thus forming the chemical scavenging sites 44, 44'. The chemical scavenger can then be deposited (e.g., via lift-off, dissolution, or another suitable technique), and the mask can be removed. In this example, the chemical scavenger sites 44, 44' can include a monolayer or thin layer of the chemical scavenger.

[0111] 2C shows a cross-sectional view of flow cell 24 including patterned opposing sequencing surfaces 32, 32′. In one example, each of these surfaces 32, 32′ can be prepared on substrate 26B, 26B′, which can then be attached to one another (e.g., via material 50) to form the example flow cell 24.

[0112] 2C, flow cell 24 includes multi-layer substrates 26B, 26B', each of which includes a support 34, 34' and a patterned material 36, 36' disposed on the support 34, 34'. The patterned material 36, 36' defines depressions 48, 48' separated by interstitial regions 46, 46'.

[0113] 2C, patterned materials 36, 36' are disposed on supports 34, 34', respectively. It should be understood that any material that can be selectively deposited, or deposited and patterned, to form recesses 48, 48' and interstitial regions 46, 46' can be used for patterned materials 36, 36'.

[0114] As an example, inorganic oxides may be selectively applied to the substrate 34, 34' via vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., TaO), aluminum oxide (e.g., AlO), silicon oxide (e.g., SiO), hafnium oxide (e.g., HfO), and the like.

[0115] As another example, the resin can be applied to the support 34, 34′ and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, droplet dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, and the like. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resin (POSS)-based resins, non-POSS epoxy resins, poly(ethylene glycol) resins, polyether resins (e.g., ring-opened epoxies), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., CYTOP® from Bellex), and combinations thereof.

[0116] As used herein, the term "polyhedral oligomeric silsesquioxane" (commercially available as POSS® from HybridPlastics) refers to a hybrid intermediate between silica (SiO2) and silicone (R2SiO) (e.g., RSiO 1.5 ) refers to a chemical composition having the chemical formula [RSiO 3 / 2 ] nwherein the R groups may be the same or different. Exemplary R groups of the polyhedral oligomeric silsesquioxanes include epoxy, azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate, and / or methacrylate, or further, for example, alkyl, aryl, alkoxy, and / or haloalkyl groups. The resin compositions disclosed herein may include one or more different cage or core structures as monomer units. The polyhedral structures may be [ka] and the like. [ka] This monomer unit typically has eight arms with functional groups R1 to R8.

[0117] The monomer unit is [ka] T such as 10 and 10 R groups, or [ka] Such as, T 12 The polyhedral oligomeric silsesquioxane-based material may alternatively comprise a T6, T14, or T16 cage structure. The average cage content may be adjusted during synthesis and / or controlled by purification methods, and a distribution of cage sizes of the monomer units may be used in the examples disclosed herein.

[0118] In some of the examples of polyhedral oligomeric silsesquioxanes disclosed herein, R1 to R8 or R 10 or R 12At least one of R1 to R8 or R 10 or R 12 may or may not be the same, and in some instances, R1 to R8 or R 10 or R 12 At least one of R1 to R8 or R 10 or R 12 At least one of the functional groups is a non-epoxy functional group. The non-epoxy functional group can be (a) a reactive group that reacts orthogonally with epoxy groups (e.g., reacts under conditions different from those of epoxy groups), i.e., a reactive group that serves as a handle for attaching the resin to an amplification primer, polymer, or polymerization agent, or (b) a group that adjusts the mechanical or functional properties of the resin, e.g., surface energy tuning. In some examples, the non-epoxy functional group is selected from the group consisting of azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, amino, hydroxyl, alkynyl, ketone, aldehyde, ester group, alkyl, aryl, alkoxy, and haloalkyl.

[0119] As shown in FIG. 2C , the patterned material 36, 36′ includes depressions 48, 48′ defined therein and interstitial regions 46, 46′ separating adjacent depressions 48, 48′, respectively. Many different layouts of the depressions 48, 48′ can be envisioned, including regular, repeating, and irregular patterns. In one example, the depressions 48, 48′ are arranged in a hexagonal grid for close packing and improved density. Other layouts can include, for example, rectilinear (rectangular) layouts, triangular layouts, etc. In some examples, the layout or pattern can be an xy format of depressions 48, 48′ in rows and columns. In other examples, the layout or pattern can be a repeating arrangement of depressions 48, 48′ and / or interstitial regions 46, 46′. In yet other examples, the layout or pattern can be a random arrangement of depressions 48, 48′ and / or interstitial regions 46, 46′. The patterns may include spots, stripes, swirls, lines, triangles, rectangles, circles, arcs, ticks, checkerboards, diagonals, arrows, squares, and / or crosshatches.

[0120] The layout or pattern of the dimples 48, 48' may be characterized by the density of the dimples 48, 48' (e.g., the number of dimples 48, 48') in a defined area. For example, the dimples 48, 48' may be spaced apart from each other by 1 mm. 2 They can be present at a density of about 2 million per mm. 2 Approximately 100 per 1mm 2 Approximately 1,000 per 1mm 2 Approximately 100,000 per mm 2 Approximately 1 million per mm 2 Approximately 2 million per 1mm 2 Approximately 5 million per mm 2 Approximately 10 million per mm 2The density of the depressions 48, 48' in the patterned material 36, 36' can be adjusted to different densities, including densities of about 50 million per 1000,000,000 or less. It should further be understood that the density of the depressions 48, 48' in the patterned material 36, 36' can be between one of the low and high values ​​selected from the ranges above. By way of example, a high-density array can be characterized as having depressions 48, 48' separated by less than about 100 nm, a medium-density array can be characterized as having depressions 48, 48' separated by about 400 nm to about 1 μm, and a low-density array can be characterized as having depressions 48, 48' separated by more than about 1 μm. While example densities are provided, it should be understood that any suitable density can be used. The density of the depressions 48, 48' can depend, in part, on the depth of the depressions 48, 48'. In some cases, it may be desirable for the spacing between the depressions 48, 48' to be even greater than the examples described herein.

[0121] The layout or pattern of the recesses 48, 48′ may also, or alternatively, be characterized in terms of average pitch, or the spacing from the center of a recess 48, 48′ to the center of an adjacent recess 48, 48′ (center-to-center spacing), or the spacing from the left edge of one recess 48, 48′ to the right edge of the adjacent recess 48, 48′ (edge-to-edge spacing). The pattern may be regular so that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch may be, for example, approximately about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, or about 100 μm. The average pitch of a particular pattern of recesses 48, 48′ may be between one of the lower and upper values ​​selected from the ranges above. In one example, the recesses 48, 48' have a pitch (center-to-center spacing) of about 1.5 μm. Although example average pitch values ​​are provided, it should be understood that other average pitch values ​​may also be used.

[0122] The size of each of the recesses 48, 48' may be characterized by its volume, open area, depth, and / or diameter.

[0123] Each of the recesses 48, 48′ can have any volume capable of confining at least some of the fluid introduced into the flow cell 24. The minimum or maximum volume can be selected to correspond, for example, to the expected throughput (e.g., multiplexing), resolution, nucleotide, or analyte reactivity for downstream use of the flow cell 24. For example, the volume can be at least about 1×10 -3 μm 3 , at least about 1 x 10 -2 μm 3 , at least about 0.1 μm 3 , at least about 1 μm 3 , at least about 10 μm 3 , at least about 100 μm 3 Alternatively or additionally, the volume may be at most about 1×10 4 μm 3 , up to about 1 × 10 3 μm 3 , up to about 100 μm 3 , up to about 10 μm 3 , up to about 1 μm 3 , up to about 0.1 μm 3 , or even less.

[0124] The area occupied by each recess opening can be selected based on criteria similar to the volume described above. For example, the area of ​​each recess opening is at least about 1×10 -3 μm 2 , at least about 1 x 10 -2 μm 2 , at least about 0.1 μm 2 , at least about 1 μm 2 , at least about 10 μm 2 , at least about 100 μm 2 Alternatively or additionally, the area may be at most about 1×10 3 μm 2 , up to about 100 μm 2 , up to about 10 μm 2 , up to about 1 μm 2 , up to about 0.1 μm 2 , up to about 1 × 10 -2 μm2 The area occupied by the opening of each recess may be greater than, less than, or in between the above values.

[0125] The depth of each of the depressions 48, 48' can be large enough to accommodate a portion of the polymer hydrogel 40, 40'. In one example, the depth can be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or additionally, the depth can be at most about 1×10 3 The depth of each of the depressions 48, 48' may be greater than, less than, or between the values ​​specified above.

[0126] In some examples, the diameter or length and width of each of the depressions 48, 48' can be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or additionally, the diameter or length and width can be at most about 1×10 3 The diameter or length and width of each of the depressions 48, 48' may be greater than, less than, or between the values ​​specified above.

[0127] In this example, at least some of the components of the sequencing surface 32, 32' may be introduced into the recesses 48, 48'. It should be understood that any space within the recesses 48, 48' that is not occupied by components of the sequencing surface 32, 32' may be considered to be part of the flow channel 28.

[0128] In the example shown in Figure 2C, a polymer hydrogel 40, 40' is disposed within each of the depressions 48, 48'. The polymer hydrogel 40, 40' may be applied as described with reference to Figure 2B, so that the polymer hydrogel 40, 40' is present in the depressions 48, 48' and not in the surrounding interstitial regions 46, 46'.

[0129] In the example shown in Figure 2C, a primer 42, 42' may be grafted onto the polymer hydrogel 40, 40' within each recess 48, 48'. The primer 42, 42' may be applied as described with reference to Figure 2B, and thus is grafted onto the polymer hydrogel 40, 40' rather than the surrounding interstitial regions 46, 46'.

[0130] 2C, the chemical capture sites 44, 44' include a chemical capture agent applied to at least some of the interstitial regions 46, 46'. For example, the chemical capture agent may be deposited into at least some of the interstitial regions 46, 46' using microcontact printing, aerosol printing, etc. to form the chemical capture sites 44, 44'. In yet another example, a mask (e.g., photoresist) may be used to define the spaces / locations where the chemical capture agent is to be deposited, thus forming the chemical capture sites 44, 44'. The chemical capture agent may then be deposited (e.g., via lift-off, dissolution, or another suitable technique), and the mask removed.

[0131] In other examples, the chemical capture sites 44, 44' comprise chemical capture agents attached to free functional groups of the polymer hydrogel 40, 40' (e.g., those not bound to the primers 42, 42'). In still other examples, the chemical capture sites 44, 44' comprise chemical capture agents attached to primers that hybridize to some of the amplification primers 42, 42'. In these examples, the chemical capture sites 44, 44' are present in the recesses 48, 48' and not in the interstitial regions 46, 46'.

[0132] Any of the examples of chemical capture agents disclosed herein can be used in the example shown in Figure 2C.

[0133] 2D shows a cross-sectional view of flow cell 24 including patterned opposing sequencing surfaces 31, 31′. In one example, each of these surfaces 31, 31′ can be prepared on substrate 26B, 26B′, which can then be attached to one another (e.g., via material 50) to form the example flow cell 24. Each of multilayer substrates 26B, 26B′ includes a support 34, 34′ and a patterned material 36, 36′ disposed on support 34, 34′. Patterned material 36, 36′ defines depressions 48, 48′ separated by interstitial regions 46, 46′.

[0134] The opposing sequencing surfaces 31, 31' do not include polymer hydrogels 40, 40' or primers 42, 42'. Rather, the opposing sequencing surfaces 31, 31' include chemical capture sites 44, 44' disposed in respective depressions 48, 48'. Each chemical capture site 44, 44' is capable of immobilizing a respective clustered solid support 13. Each clustered solid support introduces a respective cluster of template strands 64 into each of the depressions 48, 48'.

[0135] The chemical trapping sites 44, 44' in Figure 2D include any example of a chemical trapping agent described herein. In this example, the chemical trapping agent can be deposited in the recesses 48, 48' using microcontact printing, aerosol printing, or the like to form the chemical trapping sites 44, 44'. In yet another example, a mask (e.g., photoresist) can be used to block the interstitial regions 46, 46', such that the chemical trapping agent is deposited in the recesses 48, 48' but not in the interstitial regions 46, 46'. In this example, the chemical trapping agent can then be deposited (e.g., via lift-off, dissolution, or another suitable technique), and the mask removed.

[0136] Although not shown, another example of flow cell 24 combines the unpatterned surface of FIG. 2B with the capture sites 44, 44' of FIG. 2D. In this example, recessed regions 38, 38' (similar to those shown in FIG. 2B) can be coated with a chemical capture agent rather than with polymer hydrogels 40, 40' and primers 42, 42'. Thus, chemical capture sites 44, 44' can be formed along the entire channel 28 in recessed regions 38, 38'. In this example, each chemical capture site 44, 44' can immobilize clustered solid supports 13 in a random distribution along the opposing sequencing surface.

[0137] As shown in Figures 2B-2D, substrates 26A and 26A' or 26B and 26B' ​​are attached to one another so that sequencing surfaces 30 and 30' or 32 and 32' or 31 and 31' face one another with flow channel 28 defined therebetween.

[0138] Substrates 26A and 26A' or 26B and 26B' ​​may be bonded to one another at some or all of the gap regions 46, 46'. The bond formed between substrates 26A and 26A' or 26B and 26B' ​​may be a chemical bond or a mechanical bond (e.g., using fasteners).

[0139] Any suitable technique may be used to bond substrates 26A and 26A' or 26B and 26B' ​​together, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, glass frit bonding, or other methods known in the art. In one example, a spacer layer (e.g., material 50) may be used to bond substrates 26A and 26A' or 26B and 26B'. The spacer layer may be any material 50 that seals at least a portion of substrates 26A and 26A' or 26B and 26B' ​​together. In some examples, the spacer layer may be a radiation absorbing material that aids in bonding.

[0140] Methods and kits for using multiple fluids An example of a method utilizing a combination of fluids with different densities is shown in Figures 3A and 3B.

[0141] The method generally includes the steps of introducing a first fluid 52 (FIG. 3) containing a first portion of target material 11 (e.g., complexes 10A, 10B, clustered solid supports 13, etc.) into flow cell 24 to immobilize target material 11 on each of two opposing sequencing surfaces 30, 30′ or 32, 32′ or 31, 31′ of flow cell 24, thereby immobilizing at least a portion of target material 11 by capture sites 44, 44′ on one of the two opposing sequencing surfaces 30, 32 or 30′, 32′, or 31, 31′, 30 or 30′, or 32 or 32′, or 31 or 31′; and removing the first fluid and any unimmobilized target material from flow cell 24. and introducing a second fluid 54 (Figure 3B) containing a second portion of the target material 11 into the flow cell 24, thereby immobilizing at least a portion of the target material 11 by the capture sites 44, 44' on the other of the two opposing sequencing surfaces 30, 32 or 30', 32', or 31, 31', 30' or 30, or 32' or 32, or 31' or 31, wherein either the first fluid 52 has a density lower than that of the target material 11 and the second fluid 54 has a density higher than that of the target material 11, or the density of the second fluid 54 has a density lower than that of the target material 11 and the density of the first fluid 52 has a density higher than that of the target material 11.

[0142] Prior to carrying out the method illustrated in Figures 3A and 3B, a target material 11 may be prepared or obtained.

[0143] In one example, complex 10A or 10B can be prepared using a nucleic acid sample and a library preparation fluid including multiple solid supports 12, 12′. In some examples, as described with reference to FIG. 1A, each of the solid supports 12, 12′ in the library preparation fluid can have, for example, an adapter (such as adapter 18) and a transposome complex attached to the adapter. Tagging and library preparation can be performed as defined in FIG. 1A to form complex 10A. The nucleic acid sample, solid supports 12, 12′, partial Y adapter, and transposase enzyme can be contained in separate fluids until it is desired to form complex 10A. In another example, each of the solid supports 12, 12′ in the library preparation fluid can have, for example, oligonucleotides attached thereto. In some examples, PCR-free nucleotide library preparation can be performed separately from the solid supports 12, 12′, and then the prepared library fragments can be hybridized to oligonucleotides on the surface of the solid supports 12, 12′, as described with reference to FIG. 1B. Other methods of library preparation (including, for example, PCR) can also be used, provided that the fragments are denatured to single-stranded fragments before hybridizing to the oligos on the solid support 12, 12'.

[0144] In another example, the clustered solid supports 13 can be prepared by amplifying library fragments in the presence of a plurality of solid supports 12, 12' functionalized with primers 42, 42'.

[0145] The target material 11 (e.g., complex 10A or 10B, or any other solid support 12, 12′ having sequenceable fragments 14, 14′ attached thereto, or clustered solid support 13) can be divided into first and second portions. The first portion of the target material 11 can be incorporated into a first fluid 52, and the second portion of the target material 11 can be introduced into a second fluid 54.

[0146] The first and second fluids 52, 54 have different densities. In one example, the first fluid 52 has a density lower than that of the target material 11, and the second fluid 54 has a density higher than that of the target material 11. In one particular example, the first fluid 52 has a density lower than that of the solid supports 12, 12′ of the complexes 10A or 10B or the clustered solid supports 13, and the second fluid 54 has a density higher than that of the solid supports 12, 12′ of the complexes 10A or 10B or the clustered solid supports 13. In another example, the second fluid 54 has a density lower than that of the target material 11, and the first fluid 52 has a density higher than that of the target material 11. In another specific example, second fluid 54 has a density lower than the density of solid supports 12, 12' of complexes 10A or 10B or clustered solid supports 13, and first fluid 52 has a density higher than the density of solid supports 12, 12' of complexes 10A or 10B or clustered solid supports 13. Thus, the density of each of fluids 52, 54 depends on the target material 11 used. In some examples, the density of complexes 10A or 10B or clustered solid supports 13 is approximately equal to the density of solid supports 12, 12' used in complexes 10A or 10B or clustered solid supports 13, and thus, in the specific example provided, the density of each of fluids 52, 54 varies depending on the solid supports 12, 12' used in target material 11.

[0147] The density of fluids 52, 54 can be measured at the capture temperature of target material 11 (e.g., complexes 10A, 10B or clustered solid supports 13) introduced into flow cell 24. In one example, the capture temperature ranges from about 18°C ​​to about 40°C.

[0148] In one example, the density of one of the fluids 52 or 54 at the capture temperature is at least 0.1 g / cm less than the density of the target material 11 (e.g., the solid support 12, 12′ or the clustered solid support 13 of the complex 10A or 10B) at the capture temperature. 3and the density of the other of fluids 54 or 52 at the capture temperature is at least 0.1 g / cm lower than the density of target material 11 (e.g., solid support 12, 12′ or clustered solid support 13 of composite 10A or 10B) at the capture temperature. 3 In one particular example, the density of the target material (e.g., solid support 12, 12') is greater than X g / cm 3 , then the density of one of the fluids 52 or 54 at the capture temperature is X g / cm 3 ~0.1g / cm 3 and the density of the other fluid 54 or 52 at the capture temperature is X g / cm 3 +0.1g / cm 3 is.

[0149] In addition to having the respective densities, fluids 52, 54 must also be compatible with target material 11. If complexes 10A, 10B are used, fluids 52, 54 must be compatible with complexes 10A, 10B and sequencing surface 30, 30' or 32, 32' or 31, 31' so that fragments 14, 14', 14'' and primers 42, 42' are not adversely affected. If a clustered solid support 13 is used, fluids 52, 54 must be compatible with clustered solid support 13 so that template strands 64 are not adversely affected.

[0150] The low-density fluid 52 or 54 can be any aqueous buffer solution (e.g., a weak acid and one of its salts (conjugate base) or a weak base and one of its salts (conjugate acid). The salt concentration in the aqueous buffer solution can be adjusted so that the density of the low-density fluid 52 or 54 is lower than the density of the target material 11 (e.g., the density of the solid support 12, 12′ of the complex 10A, 10B or the clustered solid support 13). The greater the density difference between the target material 11 and the low-density fluid 52 or 54, the faster the settling time of the target material 11 (e.g., the complex 10A, 10B or the clustered solid support 13) in the low-density fluid 52 or 54. By way of example, the low-density fluid 52 or 54 can be a Tris-HCl buffer or a 0.5× saline sodium citrate (SSC) buffer. In one example, the low-density fluid 52 or 54 has a density of about 1 g / cm 3 The low density fluid 52 or 54 is a buffered aqueous solution having a density of about 1.18 g / cm 3 This may be particularly suitable for use with a target material 11 having a density of

[0151] The dense fluid 54 or 52 can be an aqueous salt solution. The salt selected should render the fluid 52 or 54 "heavy" and not adversely affect the target material. If complexes 10A, 10B are used, the salt should not adversely affect complexes 10A, 10B or primers 42, 42'. If a clustered solid support 13 is used, the salt should not adversely affect template strands 64. The salt concentration in the buffered aqueous solution can be adjusted so that the density of the dense fluid 54 or 52 is greater than the density of the target material 11. Examples of dense fluids 54 or 52 include sodium polytungstate solution and sodium chloride solution. In one example, the dense fluid 54 or 52 has a density of about 2 g / cm. 3 to about 3 g / cm 3 These dense fluids 54 or 52 are sodium polytungstate solutions having densities in the range of about 1.18 g / cm 3In these examples, the sodium polytungstate solution has a concentration ranging from about 1 gram of sodium polytungstate per milliliter of water to about 2.52 grams of sodium polytungstate per milliliter of water. In another example, a 25% (w / v) sodium chloride solution has a density of about 1.2 g / cm. 3 It has a density of

[0152] In one example, the first or second fluid 52 or 54 having a density lower than that of the target material is a buffered aqueous solution, and the second or first fluid 54 or 52 having a density higher than that of the target material is a sodium polytungstate solution or a sodium chloride solution. In another example, the density of the first or second fluid 52 or 54 lower than that of the target material is about 1 g / cm at the capture temperature. 3 where the density of the second or first fluid 54 or 52, which is greater than the density of the target material, is about 2 g / cm at the capture temperature. 3 is.

[0153] As shown in Figure 3A, one example of a method includes introducing a first fluid 52 containing some of the target material 11 (e.g., complexes 10A of Figure 3A) into the flow cell 24. In this example, the first fluid 52 has a density lower than the density of the solid support 12, 12' of complexes 10A, and therefore complexes 10A migrate or sediment to the lower sequencing surface 30'. Capture sites 44' (not shown in Figure 3A) immobilize at least some of complexes 10A at the lower sequencing surface 30'.

[0154] It should be understood that some complexes 10A (or other target materials 11) in first fluid 52 may not precipitate, and these complexes 10A (or other target materials) are removed from flow cell 24 before further processing. A predetermined time period can be allowed to elapse before removing first fluid 52 and any unimmobilized target materials (e.g., complexes 10A) from flow cell 24. In one example, the predetermined time period can range from about 5 minutes to about 30 minutes to obtain a desired number of immobilized complexes 10A or other target materials 11. Longer incubation times can also be used.

[0155] Next, the exemplary method includes washing the first fluid 52 and unimmobilized target material 11 (e.g., complex 10A) from the flow cell 24. Washing may include introducing a wash fluid into the flow cell 24''. The flow may not settle, but may push the immobilized complex 10A (or other target material 11) at the sequencing surface 30' through the outlet port of the flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between the complex 10A (or other target material 11) and the capture site 44' of the sequencing surface 30' may prevent the settling and immobilized complex 10A (or other immobilized target material 11) from becoming part of the outlet flow. Furthermore, the target material 11 (e.g., complex 10A in FIG. 3A) immobilized on one of the two opposing sequencing surfaces (e.g., sequencing surface 30' in FIG. 3A) remains immobilized on that sequencing surface when the second fluid 54 is introduced.

[0156] 3B, this example method includes introducing a second fluid 54 containing some other target material 11 (e.g., complexes 10A) into the flow cell 24. In this example, the second fluid 54 has a density greater than the density of the solid supports 12, 12′ of the complexes 10A (or other target materials 11), and therefore the complexes 10A migrate to the upper sequencing surface 30. Capture sites 44 (not shown in FIG. 3B) immobilize at least some of the complexes 10A at the sequencing surface 30.

[0157] Prior to performing seeding, amplification, and sequencing or sequencing (described below), this exemplary method may further include removing the second fluid 54 and unimmobilized target material 11 from the flow cell 24. Accordingly, this exemplary method may then include washing the second fluid 54 and uncaptured target material 11 (e.g., unimmobilized complexes 10A) from the flow cell 24. Washing may be performed as described herein. The flow may push any complexes 10A (or other target materials 11) that are not immobilized on the upper sequencing surface 30 through the outlet port of the flow cell 24. It should be understood that the immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between complexes 10A (or other target materials 11) and the capture sites 44, 44′ of the sequencing surfaces 30, 30′, respectively, may prevent immobilized complexes 10A (or other immobilized target materials 11) from becoming part of the outlet flow.

[0158] If complexes 10A or 10B are used, this washing step can be followed by release and amplification of the library fragments (examples of which are described with reference to Figures 9A to 9C). If clustered solid supports 13 are used, this washing step can be followed by sequencing.

[0159] 3A and 3B show the introduction of a low-density fluid followed by a high-density fluid, it should be understood that the high-density fluid can be introduced first, with target material 11 immobilized on the upper sequencing surface 30, and then the low-density fluid can be introduced, with target material 11 immobilized on the lower / bottom sequencing surface 30'. Furthermore, it should be understood that this method can be performed with any example of a flow cell 24 disclosed herein, including those having patterned surfaces 32, 32'. When a clustered solid support 13 is used, a flow cell 24 without amplification primers 42, 42', as shown and described with reference to FIG. 2D, can be used.

[0160] 3A and 3B may include a preparation fluid containing target material 11, a first introduction fluid (e.g., fluid 52 or 54) having a density lower than that of target material 11, and a second introduction fluid (fluid 54 or 52) having a density higher than that of target material 11. In one example kit, the first introduction fluid is a buffered aqueous solution, and the second introduction fluid is a sodium polytungstate solution or a sodium chloride solution. In one example where the second introduction fluid is a sodium polytungstate solution, the sodium polytungstate solution has a concentration of about 1 gram of sodium polytungstate per milliliter of water. In another example kit, the density of the first introduction fluid at the capture temperature is at least 0.1 g / cm 3 higher than the density of target material 11 at the capture temperature. 3 and the density of the second introduced fluid at the capture temperature is at least 0.1 g / cm 3 lower than the density of the target material 11 at the capture temperature. 3 In yet another example, the density of the first introduction fluid is about 1 g / cm at the trapping temperature. 3 and the density of the second introduced fluid is about 2 g / cm at the trapping temperature. 3 is.

[0161] In some examples, the preparation fluid containing target material 11 includes solid supports 12, 12', and the kit may also include other library preparation components such as nucleic acid samples, partial Y adaptors, transposase enzymes, etc., each of which may be contained in a separate fluid until it is desired to form target material 11, such as complexes 10A, 10B, clustered solid supports 13, etc. Some examples of kits may also include a flow cell 24. Other examples of kits may include a preparation fluid containing any example of target material 11 disclosed herein.

[0162] One-Fluid Method and Kit Other examples of the methods disclosed herein utilize one fluid during immobilization of the target material 11. Some methods utilize one target material 11 and different modalities to achieve immobilization across opposing sequencing surfaces 30, 30' or 32, 32' or 31, 31'. Other methods utilize two different target materials 11 (each having at least one property different from the other) and the same or different modalities to achieve immobilization across opposing sequencing surfaces 30, 30' or 32, 32' or 31, 31'. Different examples are described herein with reference to Figures 4A and 4B through 8A and 8B.

[0163] Prior to carrying out any of the methods shown in Figures 4A and 4B through 8A and 8B, complexes 10A or 10B or clustered solid supports 13 may be prepared as described herein.

[0164] Complex 10A or 10B can be prepared using a nucleic acid sample and a library preparation fluid including a plurality of magnetic solid supports 12′. In some examples, as described with reference to FIG. 1A, each of the magnetic solid supports 12′ in the library preparation fluid can have, for example, an adapter (such as adapter 18) and a transposome complex attached to the adapter. Tagging and library preparation can be performed as defined in FIG. 1A to form complex 10A. The nucleic acid sample, magnetic solid support 12′, partial Y adapter, and transposase enzyme can be contained in separate fluids until it is desired to form complex 10A. In other examples, each of the magnetic solid supports 12′ in the library preparation fluid can have, for example, an oligonucleotide attached thereto. In some examples, a PCR-free nucleotide library preparation can be performed separately from the magnetic solid supports 12′, after which the prepared library fragments can be hybridized to oligonucleotides on the surface of the magnetic solid supports 12′, as described with reference to FIG. 1B. Other methods of library preparation (including, for example, PCR) can also be used, provided that the fragments are denatured to single-stranded fragments before hybridizing to the oligos on the magnetic solid support 12'.

[0165] The clustered solid supports 13 can be prepared by amplifying library fragments in the presence of a plurality of solid supports 12, 12' functionalized with primers 42, 42'.

[0166] An example of a method utilizing a fluid, a substantially uniform magnetic force, and a magnetically responsive target material, such as a solid support 12′, is shown in Figures 4A and 4B. The method generally includes the steps of immobilizing target material 11 on each of two opposing sequencing surfaces 30, 30′ or 32, 32′ of flow cell 24 by introducing a fluid 56 containing target material 11 into flow cell 24, where fluid 56 has a density approximately equal to that of magnetic solid support 12′; allowing a portion of target material 11 to be immobilized by capture sites 44 or 44′ (not shown in Figure 4A) on one of two opposing sequencing surfaces 30, 30′, 32, 32′, or 31, 31′; and and applying a magnetic force to the other of the opposing sequencing surfaces 30, 30', or 30, or 32' or 32, or 31' or 31, thereby drawing other ones of the target materials 11 to the other of the two opposing sequencing surfaces 30, 30', or 32, 32', or 31, 31', or 30' or 30, or 32' or 32, or 31' or 31, where they are immobilized by capture sites 44' or 44 (not shown in FIG. 4B ) of one of the other of the two opposing sequencing surfaces 30, 30', or 32, 32', or 31, 31'. If complexes 10A, 10B are used, this exemplary method may further include ceasing application of the magnetic force and removing fluid and unimmobilized target material from flow cell 24 before performing seeding and amplification (as described below). These steps may be followed by release and amplification of the library fragments (eg, as described with reference to Figures 9A-9C).

[0167] Target materials 11 (e.g., complexes 10A, 10B, or any other magnetic solid support 12' having sequenceable fragments 14, 14', 14" or clustered solid supports 13) can be incorporated into fluid 56. As an example, from about 25,000 target materials 11 (e.g., complexes 10A, 10B or clustered solid supports 13) to about 500,000 target materials 11 can be included in 1 microliter of fluid. As another example, from about 100,000 target materials 11 to about 500,000 target materials 11 can be included in 1 microliter of fluid. Other concentrations can be used depending on the size of flow cell 24.

[0168] The density of the fluid 56 can be measured at the trapping temperature of the target material 11 introduced into the flow cell 24. In one example, the trapping temperature ranges from about 18°C ​​to about 40°C.

[0169] Fluid 56 is selected to have a density that is at least approximately equal to the density of magnetic solid support 12' of target material 11. In these examples, "at least approximately equal" means that the density of fluid 56 is less than 0.08 g / cm of the density of magnetic solid support 12'. 3 This means that the fluid 56 and the magnetic solid support 12' have the same density. By having a density at least approximately equal to that of the magnetic solid support 12', the fluid 56 functions as a gentle flotation agent. As used herein, the term "gentle flotation agent" refers to a fluid in which the target material 11 (e.g., complexes 10A, 10B, clustered solid supports 13, etc.) can remain suspended for at least some period of time before sinking or settling. In the fluid 56, some of the target material 11 begins to sink and become immobilized on the lower / bottom sequencing surface 30', 32', 31' within the flow cell 24, while other target material 11 remains floating (at least for some period of time).

[0170] Fluid 56 can be any buffered aqueous solution. The salt concentration in the buffered aqueous solution can be adjusted so that the density of fluid 56 is at least approximately equal to the density of magnetic solid support 12'. In other words, the salt concentration in the buffered aqueous solution can be adjusted so that the density of fluid 56 is at least approximately equal to the density of magnetic solid support 12' + / - 0.08 g / cm. 3 The density of the magnetic solid support 12' and the fluid 56 can be adjusted to be within 1.1 g / cm. For example, the fluid 56 can be a Tris-HCl buffer, a 0.5x saline sodium citrate (SSC) buffer, or a 75 mM sodium citrate solution (pH=7) containing about 750 mM NaCl. In one example, the density of each of the magnetic solid support 12' and the fluid 56 can be about 1.1 g / cm. 3 is.

[0171] After fluid 56 and target material 11 are introduced into flow cell 24, target material 11 initially floats within fluid 56. Over time, some of the target material 11 settles to lower / lower sequencing surfaces 30', 32', 31', where it becomes immobilized at capture sites 44'. An example is shown in FIG. 4A, where some of complex 10A settles to lower / lower sequencing surface 30'. Fluid 56 helps prevent all of the target material 11 from settling too quickly on lower / lower sequencing surfaces 30', 32', 31'.

[0172] Thus, after the introduction of fluid 56 and immobilization of a portion of target material 11, there is a time during which an externally applied magnetic force is applied to the other sequencing surfaces 30, 32, 31 within flow cell 24. The magnetic force attracts the suspended target material 11 to the upper / top sequencing surfaces 30, 32, 31 of flow cell 24. An example of a portion of complex 10A migrating to the upper / top sequencing surface 30 is shown in Figure 4B.

[0173] In this exemplary method, a predetermined time may elapse between the introduction of fluid 56 and the application of magnetic force. This time lapse may be desirable to allow a portion of target material 11 to settle and become immobilized on one sequencing surface 30', 32', 31', while the remaining target material 11 floats within fluid 56. In one example, this predetermined time period ranges from about 5 minutes to about 30 minutes. In some examples, a predetermined time period elapses between the introduction of fluid 56 and the application of magnetic force, and the predetermined time period ranges from about 5 seconds to about 2 minutes.

[0174] Next, as shown in FIG. 4B , a magnetic force is applied by placing a magnet 58 on the exterior surface 60 of the flow cell 24 adjacent to the sequencing surfaces 30, 32. The magnet 58 should have a magnetic field strength sufficient to attract the suspended target material 11 (e.g., complexes 10A, 10B, clustered solid supports 13, etc.) without attracting target material 11 already immobilized on the lower / lower sequencing surfaces 30′, 32′, 31′. The magnetic field strength is relatively weak but is applied at least substantially uniformly across the entire length and width of the flow channel 28. The relatively weak magnetic field strength can range from about 1 mT (milliTesla) to about 100 mT. In some examples, the strength of the relatively weak magnetic field ranges from about 1 mT to about 10 mT, or from about 10 mT to about 100 mT. This can immobilize the suspended target material 11 and capture sites 44 across the upper / upper sequencing surfaces 30, 32, 31. Stronger magnets such as neodymium magnets can be used, the field strength of these magnets being around 1 T (tesla).

[0175] In one example, the magnet 58 has the same length and width as the flow channel 28 and / or flow cell 24. In one example, the magnet 58 is similar to a refrigerator magnet and has a magnetic field strength of approximately 5 mT. In another example, the magnet 58 is an elastomeric strip with embedded small magnetic particles. These types of flexible magnets are commercially available from, for example, Uline, Arnold Magnetic Technologies (FLEXMAG™), and others. In one example, applying the magnetic force involves placing an elastomeric strip with embedded magnetic particles on the outer surface 60 of the flow cell 24 adjacent to the other of two opposing sequencing surfaces (i.e., the sequencing surface 30 on which the target material 11 is not immobilized). In some examples, the magnet can be applied manually. In other examples, applying the magnetic force can be automated, for example, when integrated into a sequencing system.

[0176] The time frame for applying the magnet 58 (and therefore the magnetic force) will vary, in part, depending on the strength of the magnet and the concentration of the complexes 10A, 10B in the fluid 56. By way of example, the magnet 58 may be applied for 5 seconds to about 2 minutes. The exemplary method then includes ceasing the application of the magnetic force, which may be accomplished by removing the magnet 58.

[0177] It should be understood that some target material 11 (e.g., complexes 10A, 10B, clustered solid supports 13) within fluid 56 may not be immobilized on any of sequencing surfaces 30, 30′ or 32, 32′ or 31, 31′, and such target material 11 may be removed from flow cell 24 prior to further processing. Thus, this exemplary method may include washing fluid 56 and uncaptured target material 11 from flow cell 24. Washing may include introducing a washing fluid into flow cell 24″. The flow may push any target material 11 that is not immobilized on sequencing surfaces 30, 30′ or 32, 32′ or 31, 31′ out through an exit port of flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between the target material 11 and the capture sites 44, 44' of the sequencing surface 30, 30' or 32, 32' or 31, 31' may prevent the immobilized target material 11 from becoming part of the outlet flow.

[0178] 4A and 4B show a flow cell 24 having sequencing surfaces 30 and 30', it should be understood that the method can be practiced with any example of a flow cell 24 disclosed herein, including those having patterned sequencing surfaces 32, 32'. When a clustered solid support 13 including magnetically responsive solid supports 12' is used, a flow cell 24 that does not include amplification primers 42, 42', such as that shown and described with reference to FIG. 2D, can be used. Additionally, other magnetically responsive target materials can be used in this example method.

[0179] 4A and 4B may include a preparation fluid containing a plurality of magnetic solid supports 12′ and an input fluid (e.g., fluid 56) having a density approximately equal to that of the magnetic solid supports 12′. The kit may also include other library preparation components, such as nucleic acid samples, partial Y adapters, and transposase enzymes, each of which may be contained in separate fluids until desired formation of target material 11, such as complexes 10A, 10B, clustered solid supports 13, etc. Some examples of the kit may also include a flow cell 24. Still other examples of the kit may include an amplification mixture containing a temperature-responsive material in liquid form.

[0180] The methods illustrated in Figures 5A and 5B, 6A and 6B, 7A and 7B, and 8A and 8B are now described. Each of these methods utilizes a combination of target materials (e.g., 11A and 11B, or 11C and 11D, etc.), with different target material combinations being described in more detail for each set of figures. Each set of figures illustrates a method performed with a flow cell 24 having an unpatterned sequencing surface 30, 30'. It should be further understood that any of these methods can be performed with any example of a flow cell 24 disclosed herein, including those having patterned surfaces 32, 32'. Furthermore, when clustered solid supports 13 are used as target materials (e.g., 11A and 11B, etc.), a flow cell 24 without amplification primers 42, 42', as shown and described with reference to Figure 2D, can be used.

[0181] An example of a method utilizing a combination of target materials 11A, 11B is shown in Figures 5A and 5B. In this example, the target materials 11A, 11B have different densities from each other and from the carrier fluid.

[0182] This exemplary method generally includes the steps of introducing a target fluid 56' containing a first target material 11A and a second target material 11B into a flow cell 24 to simultaneously immobilize the first target material 11A on a first surface 30 or 32 or 31 of two opposing sequencing surfaces 30, 30', or 32, 32', or 31, 31' of the flow cell 24, and the second target material 11B on a second surface 30' or 32' or 31' of the two opposing sequencing surfaces 30, 30', or 32, 32', or 31, 31' of the flow cell 24, wherein the carrier fluid of the target fluid 56' has a fluid density, the first target material 11A has a first density lower than the fluid density, and the second target material 11B has a second density higher than the fluid density.

[0183] The density of the carrier fluid of the target fluid 56' can be measured at the capture temperature of the target material 11A, 11B introduced into the flow cell 24. In one example, the capture temperature ranges from about 18°C ​​to about 40°C.

[0184] In one example, the density of one of the target materials 11A is at least 0.1 g / cm 3 greater than the density of the carrier fluid at the capture temperature. 3 and the density of the other target material 11B is at least 0.1 g / cm 3 lower than the density of the carrier fluid at the capture temperature. 3 In one particular example, the density of the carrier fluid at the trapping temperature is X g / cm 3 , the density of one of the target materials 11A or 11B is X g / cm 3 ~0.1g / cm 3 and the density of the other of the target materials 11B or 11A is X g / cm 3 +0.1g / cm 3 is.

[0185] The carrier fluid of the target fluid 56' can be any of the aqueous buffer solutions or salt solutions described herein. The salt concentration in the aqueous buffer solution or salt solution can be adjusted so that the density of the carrier fluid at the capture temperature is between the densities of the target materials 11A, 11B, respectively. In another example, the carrier fluid of the target fluid 56' is an ionic liquid.

[0186] The target materials 11A, 11B can be complexes 10A, 10B or clustered solid supports 13. The supports 12 for the target materials 11A, 11B can be any of the embodiments described herein, provided that the densities of the respective materials 11A, 11B are different with respect to the carrier fluid, as described in this exemplary method. The density of the solid support 12 for each of the target materials 11A, 11B is at least approximately equal to the density of the respective target material 11A, 11B. Thus, the solid support 12 for the target material 11A is selected to have a density lower than the density of the carrier fluid for the target fluid 56' at the capture temperature, and the solid support 12 for the target material 11B is selected to have a density higher than the density of the carrier fluid for the target fluid 56' at the capture temperature.

[0187] 5A, the method includes introducing a target fluid 56' containing target materials 11A, 11B into a flow cell 24. The target fluid 56' is allowed to incubate within the flow cell 24 for a predetermined period of time. In one example, the predetermined period of time may range from about 5 minutes to about 30 minutes to obtain a desired number of immobilized target materials 11A, 11B on the sequencing surface 30, 30'. Longer incubation times may also be used.

[0188] As previously mentioned, as shown in FIG. 5B, the solid support 12 of target material 11A has a density lower than that of the carrier fluid at the capture temperature, and therefore, target material 11A migrates or floats to the upper sequencing surface 30. Capture sites 44 (not shown in FIG. 5B) immobilize at least a portion of target material 11A to the upper sequencing surface 30. Also, as previously mentioned, as shown in FIG. 5B, the solid support 12 of target material 11B has a density higher than that of the carrier fluid at the capture temperature, and therefore, target material 11B migrates or sinks to the lower sequencing surface 30'. Capture sites 44' (also not shown in FIG. 5B) immobilize at least a portion of target material 11B to the lower / bottom sequencing surface 30'.

[0189] Immobilization of the target materials 11A, 11B occurs simultaneously with introduction of the target fluid 56' into the flow cell 24 due to the different densities of the target materials 11A, 11B relative to the carrier fluid. Thus, in the method of Figures 5A and 5B, at least a portion of the first target material 11A is immobilized by respective capture sites 44' on the first surfaces of the two opposing sequencing surfaces 30, and at least a portion of the second target material 11B is immobilized by respective capture sites 44' on the second surfaces of the two opposing sequencing surfaces 30'.

[0190] It should be understood that some target materials 11A, 11B may not be immobilized, and such target materials 11A, 11B are removed from the flow cell 24 before further processing. Therefore, this exemplary method then includes washing the carrier fluid of the target fluid 56′ and any unimmobilized target materials 11A, 11B from the flow cell 24. Washing may include introducing a wash fluid into the flow cell 24″. The flow may push any target materials 11A, 11B that are not immobilized on the sequencing surface 30, 30′ through the outlet port of the flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between each target material 11A, 11B and the capture sites 44, 44′ of the sequencing surface 30, 30′ may prevent the immobilized target materials 11A, 11B from becoming part of the outlet flow.

[0191] If complexes 10A or 10B are used as target material 11A, 11B, this washing step may be followed by release and amplification of the library fragments (examples of which are described with reference to Figures 9A to 9C). If clustered solid supports 13 are used, this washing step can be followed by sequencing.

[0192] A kit for carrying out the method described with reference to Figures 5A and 5B may include a target fluid 56' including a carrier fluid having a fluid density, a first target material 11A having a first density lower than the fluid density, and a second target material 11B having a second density higher than the fluid density.

[0193] In some examples, the first and second target materials 11A, 11B are complexes 10A or 10B. In these examples, the first target material 11A includes a first solid support 12 having a first solid support density approximately equal to the first density (i.e., lower than the fluid density) and sequenceable nucleic acid fragments 14, 14', 14" attached to the first solid support 12, and the second target material 11B includes a second solid support 12 having a second solid support density approximately equal to the second density (i.e., higher than the fluid density) and sequenceable nucleic acid fragments 14, 14', 14" attached to the second solid support 12.

[0194] In other examples, the first and second target materials 11A, 11B are clustered solid supports 13. In these examples, the first target material 11A includes a first solid support 12 having a first solid support density approximately equal to the first density (i.e., less than the fluid density) and a first cluster of template strands 64 attached to the first solid support 12, and the second target material 11B includes a second solid support 12 having a second solid support density approximately equal to the second density (i.e., greater than the fluid density) and a second cluster of template strands 64 attached to the second solid support 12.

[0195] Alternatively, the kit may include a carrier fluid, reagents, and materials for preparing target material 11 A, and reagents and materials for preparing target material 11 B. In this example, each target material 11 A, 11 B may be prepared as described herein using the respective reagents and materials, which may then be added to a carrier fluid to form target fluid 56′.

[0196] Other examples of this method utilize different target materials and different modalities to immobilize the target materials. These examples generally involve introducing first and second target materials into a flow cell 24 including two opposing sequencing surfaces 30, 30', or 32, 32', or 31, 31', where the first target material has at least one property different from the second target material, the at least one property being selected from the group consisting of density, charge, magnetism, and combinations thereof, and exposing the first and second target materials to at least one condition such that the first target material is immobilized by a first capture site 44 of the two opposing sequencing surfaces 30, 32, or 31, and the second target material is immobilized by a second capture site 44' of the two opposing sequencing surfaces 30', 32', 31'.

[0197] An example method is shown in Figures 6A and 6B. In this example, target materials 11C, 11D have opposite charges.

[0198] As shown in Figure 6A, first target material 11C has a negative charge, and second target material 11D has a positive charge. Any example of charged solid support 12 described herein can be used in this embodiment. In one example, negatively charged first target material 11C is selected from the group consisting of carboxylated solid supports, polyglutamic acid-coated solid supports, and sulfate-functionalized solid supports, and positively charged second target material 11D is selected from the group consisting of amine-functionalized solid supports, such as chitosan-functionalized solid supports and polylysine-functionalized solid supports.

[0199] The target materials 11C, 11D may be part of a fluid 56'' introduced into the flow cell 24. In this example, the fluid 56'' used to introduce the charged target materials 11C, 11D into the flow cell 24 may be an electrolyte. As an example, the fluid 56'' may be a combination of tris(hydroxymethylaminomethane) and boric acid present at the same molar concentration (e.g., 4.5 mM each). When complexes 10A, 10B are used as target materials 11C, 11D, approximately 4 mM Mg 2+A low-salt buffer such as saline-sodium citrate (SSC) buffer (e.g., about 45 mM) containing HCl can be used. This type of fluid 56" maximizes the charge on the charged target materials 11C, 11D while allowing hybridization of the library fragments 14, 14', 14' as they are released. When clustered solid supports 13 are used as target materials 11C, 11D, water can be used as fluid 56".

[0200] Additionally, the densities of the fluid 56'' and the target materials 11C, 11D may be approximately equal such that the densities of the target materials 11C, 11D do not interfere with the electrostatically induced movement of the target materials 11C, 11D. In another example, the densities of the fluid 56'' and the target materials 11C, 11D may not be equal. In this example, the force due to the applied electric field 62 is greater than any force due to the density difference.

[0201] In this example method, the conditions to which the charged target materials 11C, 11D are exposed to initiate simultaneous migration and immobilization are an electric field 62 applied between two opposing sequencing surfaces 30 and 30', 32 and 32', or 31 and 31' to generate positive charges 66 on a first surface of the two opposing sequencing surfaces 30, 32, 31 and negative charges 68 on a second surface of the two opposing sequencing surfaces 30', 32', 31'.

[0202] To generate an electric field 62 across the flow cell 24, each sequencing surface 30, 30' or 32, 32' or 31, 31' may be electrically connected to a power source to produce respective charges 66, 68 that attract the respective target materials 11C, 11D. In the example shown in Figures 6A and 6B, the electric field 62 is applied in a direction toward the lower / bottom sequencing surface 30', resulting in the upper sequencing surface 30 being positively charged and the lower / bottom sequencing surface 30' being negatively charged.

[0203] Immobilization of target materials 11C, 11D occurs simultaneously with exposure of fluid 56'' within flow cell 24 to electric field 62. This is due to the positive and negative charges of target materials 11C, 11D and their respective responses to the applied electric field 62. Negatively charged target material 11C migrates toward the now positively charged sequencing surface 30, where it is immobilized by capture sites 44 (not shown in FIG. 6B) on the upper sequencing surface 30. Positively charged target material 11D migrates toward the now negatively charged sequencing surface 30', where it is immobilized by capture sites 44 (not shown in FIG. 6B) on the lower / bottom sequencing surface 30'.

[0204] The electric field 62 can be applied for a predetermined time. In one example, the predetermined time can range from about 1 minute to about 30 minutes to obtain a desired number of immobilized target materials 11C, 11D on each sequencing surface 30, 30′. In other examples, the electric field 62 can be applied for a time ranging from about 1 minute to about 2 minutes, or from about 1 minute to about 5 minutes, or from about 5 minutes to about 30 minutes, etc.

[0205] It should be understood that some target materials 11C, 11D may not be immobilized, and such target materials 11C, 11D are removed from the flow cell 24 before further processing. The electric field 62 may be stopped before removing the non-immobilized target materials 11C, 11D. Thus, this exemplary method may include removing the electric field 62 and then washing the fluid 56″ and non-immobilized target materials 11C, 11D from the flow cell 24. Washing may include introducing a washing fluid into the flow cell 24″. The flow may push any target materials 11C, 11D that are not immobilized on the sequencing surface 30, 30′ through the outlet port of the flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between each target material 11C, 11D and the capture sites 44, 44′ of the sequencing surface 30, 30′ may prevent the immobilized target materials 11C, 11D from becoming part of the outlet flow.

[0206] If complexes 10A or 10B are used as target material 11C, 11D, this washing step may be followed by release and amplification of the library fragments (for example, examples of which are described with reference to Figures 9A to 9C). If clustered solid supports 13 are used, this washing step can be followed by sequencing.

[0207] Another exemplary method is shown in Figures 7A and 7B. In this example, the target materials 11E, 11F have different magnetic properties and densities.

[0208] In this example (as shown in FIG. 7A), target materials 11E, 11F are introduced into flow cell 24 in a fluid 56''' having a first density. As explained in more detail below, the density of each of target materials 11E, 11F is selected for this first density, i.e., fluid 56''', at the capture temperature of target materials 11E, 11F. The capture temperature ranges from about 18°C ​​to about 40°C.

[0209] In the example shown in Figures 7A and 7B, the first target material 11E is magnetic and the second target material 11F is non-magnetic and has a density higher than the first density (i.e., the density of the fluid 56''' at the capture temperature).

[0210] In this example, the first target material 11E comprises any of the magnetically responsive solid supports 12' disclosed herein. Furthermore, the densities of the fluid 56'" and the target material 11E can be approximately similar so that the density of the target material 11E does not interfere with the magnetically induced movement of the target material 11E. In another example, the densities of the fluid 56'" and the target material 11E may not be similar. In this example, the magnitude of the force due to the applied electric field 70 is greater than any force due to the difference in density.

[0211] Also in this example, second target material 11F comprises any of the solid supports 12 disclosed herein that are magnetically unresponsive. The density of solid support 12, and therefore target material 11F, is greater than the density of fluid 56'" at the capture temperature. Therefore, target material 11F is unresponsive to an applied magnetic field and, because it is heavier than fluid 56'", is able to migrate or settle to lower sequencing surface 30'.

[0212] In this example method, fluid 56''' containing target materials 11E, 11F is introduced into flow cell 24 (FIG. 7A), and the condition to which target materials 11E, 11F are exposed to initiate simultaneous migration and immobilization is the application of magnetic force 70 (FIG. 7B). The density of fluid 56''' can also be considered a condition that affects migration and immobilization.

[0213] A magnetic force (or magnetic field 70, as shown in FIG. 7B) can be applied as described with reference to FIGS. 4A and 4B. In the example shown in FIG. 7B, the magnetic force / magnetic field 70 is applied in the direction of the upper sequencing surface 30, resulting in the magnetically responsive (first) target material 11E migrating in the same direction toward the upper sequencing surface 30. Capture sites 44 (not shown in FIG. 7A or 7B) immobilize at least a portion of the target material 11E on the upper sequencing surface 30. At the same time, the solid support 12 of the target material 11F is magnetically unresponsive and is heavier than the fluid 56''' at the capture temperature. Thus, as shown in FIG. 7B, the target material 11F migrates to or settles onto the lower sequencing surface 30'. Capture sites 44' (not shown in FIG. 7A or 7B) immobilize at least a portion of the target material 11F on the lower / bottom sequencing surface 30'.

[0214] The magnetic force / field 70 may be applied for a predetermined time period, which in one example may range from about 5 minutes to about 30 minutes to obtain a desired number of immobilized target materials 11E on the sequencing surface 30.

[0215] Immobilization of target materials 11E, 11F occurs upon introduction of target fluid 56''' into flow cell 24 and upon exposure to magnetic field 70 due to the properties (both density and magnetic properties) of target materials 11E, 11F. In the method of Figures 7A and 7B, at least some of first target material 11E is immobilized by respective capture sites 44 on a first surface of two opposing sequencing surfaces 30, and at least some of second target material 11F is immobilized by respective capture sites 44' on a second surface of two opposing sequencing surfaces 30'.

[0216] It should be understood that some target materials 11E, 11F may not be immobilized, and such target materials 11E, 11F are removed from the flow cell 24 before further processing. The magnetic force / field 70 may be stopped prior to removing the non-immobilized target materials 11E, 11F. Thus, this exemplary method may include removing the magnetic force / field 70 and then washing the fluid 56''' and non-immobilized target materials 11E, 11F from the flow cell 24. Washing may include introducing a washing fluid into the flow cell 24''. The flow may push any target materials 11E, 11F that are not immobilized on the sequencing surface 30, 30' through the outlet port of the flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between each target material 11E, 11F and the capture sites 44, 44' of the sequencing surface 30, 30' may prevent the immobilized target materials 11E, 11F from becoming part of the outlet flow.

[0217] If complexes 10A or 10B are used as target material 11E, 11F, this washing step may be followed by release and amplification of the library fragments (examples of which are described with reference to Figures 9A to 9C). If clustered solid supports 13 are used as target material 11E, 11F, this washing step may be followed by sequencing.

[0218] 7A and 7B can also be performed such that magnetically responsive target material 11E is immobilized on the lower / bottom sequencing surface 30' and non-magnetically responsive target material 11F is immobilized on the upper sequencing surface 30. In this example, the non-magnetically responsive target material 11F comprises a solid support 12 selected to have a density lower than that of the fluid 56''' at the capture temperature. In this example, target material 11E responds to a magnetic force / field (applied in the direction of the lower sequencing surface 30') and is attracted to the lower sequencing surface 30', while target material 11F does not respond to the applied magnetic field and is lighter than the fluid 56''' and is therefore able to float or move to the upper sequencing surface 30.

[0219] Another exemplary method is shown in Figures 8A and 8B. In this example, target materials 11G, 11H have different charges and densities.

[0220] In this example, target materials 11G, 11H are introduced into flow cell 24 in a fluid 56'''' having a first density. As explained in more detail below, the density of each of target materials 11G, 11H is selected with respect to this first density, i.e., the density of fluid 56'''' at the capture temperature of target materials 11G, 11H. The capture temperature ranges from about 18°C ​​to about 40°C.

[0221] In these examples, the fluid 56'''' is an electrolyte.

[0222] In the example shown in FIGS. 8A and 8B, the first target material 11G is negatively charged, and the second target material 11H is neutral (uncharged) and has a density greater than the first density (i.e., the density of the fluid 56"" at the capture temperature). In this example, the first target material 11G comprises any of the negatively charged solid supports disclosed herein, such as a carboxylated solid support, a polyglutamic acid-coated solid support, or a sulfate-functionalized solid support. Furthermore, the density of the fluid 56"" and the density of the target material 11G can be approximately equal so that the density of the target material 11G does not interfere with electrostatically induced migration of the negatively charged target material 11G. Alternatively, the density of the target material 11G can be lower than the density of the fluid 56"", and both the density and charge can aid in the migration of the target material 11G.

[0223] In another example of the method represented by FIGS. 8A and 8B, the first target material 11G is positively charged, and the second target material 11H is neutral (uncharged) and has a density greater than the first density (i.e., the density of the fluid 56''' at the capture temperature). In this example, the first target material 11G comprises any of the positively charged solid supports disclosed herein, such as an amine-functionalized solid support (e.g., chitosan or a polylysine-functionalized solid support). Furthermore, the density of the fluid 56'''' and the density of the target material 11G can be approximately equal so that the density of the target material 11G does not interfere with electrostatically induced migration of the positively charged target material 11G. Alternatively, the density of the target material 11G can be lower than the density of the fluid 56'''', and both the density and charge can aid in the migration of the target material 11G.

[0224] 8A and 8B, the second target material 11H comprises any of the solid supports 12 disclosed herein that are uncharged. The density of the solid support 12, and therefore the target material 11H, is greater than the density of the fluid 56'''' at the capture temperature. Thus, the target material 11H does not respond to the applied electric field 62 and, because it is heavier than the fluid 56'', can migrate or settle to the lower sequencing surface 30'.

[0225] Fluid 56'''' containing target materials 11G, 11H is introduced into flow cell 24, and the condition to which target materials 11G, 11H are exposed to initiate simultaneous migration and immobilization is the application of electric field 62. The density of fluid 56'''' can also be considered a condition that affects migration and immobilization.

[0226] The electric field 62 can be applied as described with reference to FIGS. 6A and 6B. In the example shown in FIG. 8A (where the target material 11G is negatively charged), the electric field 62 is applied in a direction toward the lower / lower sequencing surface 30′, thereby causing the upper sequencing surface 30 to become positively charged and the lower / lower sequencing surface 30′ to become negatively charged. In this example, the negatively charged target material 11G migrates toward the now positively charged sequencing surface 30, where it is immobilized by the capture sites 44 (not shown in FIG. 8A or 8B) of the upper sequencing surface 30. At the same time, the solid support 12 of the target material 11H is uncharged and is heavier than the fluid 56″″ at the capture temperature. Therefore, the target material 11H migrates to or settles onto the lower sequencing surface 30′, as shown in FIG. 8B. Capture sites 44' (not shown in either Figure 8A or Figure 8B) immobilize at least a portion of the target material 11H to the lower / bottom sequencing surface 30'.

[0227] As noted above, in another example of the method represented by FIGS. 8A and 8B, target material 11G is positively charged. In this example, electric field 62 is applied in a direction toward upper sequencing surface 30 (i.e., in a direction opposite to that shown in FIGS. 8A and 8B). This causes lower sequencing surface 30′ to become positively charged and upper sequencing surface 30 to become negatively charged. In this example, positively charged target material 11G migrates toward the now negatively charged upper sequencing surface 30, where it is immobilized by capture sites 44 on upper sequencing surface 30. Simultaneously, solid support 12 of target material 11H is uncharged and is heavier than fluid 56″″ at the capture temperature. Thus, similar to FIG. 8B, target material 11H migrates to or settles onto lower sequencing surface 30′. Capture sites 44′ (also not shown in FIG. 8B) immobilize at least a portion of target material 11H to lower / bottom sequencing surface 30′.

[0228] 8A and 8B, the electric field 62 can be applied for a predetermined time period, which in one example can range from about 1 minute to about 30 minutes to obtain a desired number of immobilized charged target materials 11G on the oppositely charged sequencing surface 30 or 30′.

[0229] Immobilization of target materials 11G, 11H occurs upon introduction of target fluid 56'''' into flow cell 24 and exposure to electric field 62 due to the properties (both density and magnetic properties) of target materials 11G, 11H. In the method of Figures 8A and 8B, at least some of first target material 11G is immobilized by respective capture sites 44 on a first surface of two opposing sequencing surfaces 30, and at least some of second target material 11H is immobilized by respective capture sites 44' on a second surface of two opposing sequencing surfaces 30'.

[0230] It should be understood that some target materials 11G, 11H may not be immobilized, and such target materials 11G, 11H are removed from the flow cell 24 before further processing. The electric field 62 may be stopped before removing the non-immobilized target materials 11G, 11H. Thus, this exemplary method may include removing the electric field 62 and then washing the fluid 56'''' and non-immobilized target materials 11G, 11H from the flow cell 24. Washing may include introducing a washing fluid into the flow cell 24''. The flow may push any target materials 11G, 11H that are not immobilized on the sequencing surface 30, 30' through the outlet port of the flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between each target material 11G, 11H and the capture sites 44, 44' of the sequencing surface 30, 30' may prevent the immobilized target materials 11G, 11H from becoming part of the outlet flow.

[0231] If complexes 10A or 10B are used as target materials 11G, 11H, this washing step may be followed by release and amplification of the library fragments (examples of which are described with reference to Figures 9A to 9C). If clustered solid supports 13 are used as target materials 11G, 11H, this washing step may be followed by sequencing.

[0232] The exemplary method shown in FIGS. 8A and 8B can also be performed such that target material 11G is uncharged and has a density lower than that of target fluid 56''''. In this example, target material 11H is positively charged. In this example, positively charged target material 11H responds to electric field 62 (applied in the direction of lower sequencing surface 30') and is attracted to lower sequencing surface 30'. Also, in this example, target material 11G does not respond to the applied magnetic field and is lighter than fluid 56''', allowing it to float or move to upper sequencing surface 30.

[0233] It should be understood that other orthogonal modalities can be combined to immobilize two different target materials 11. Each of the target materials 11 may respond to one of the orthogonal modalities but not the other, allowing the modalities to independently affect one of the target materials 11. For example, an uncharged magnetically responsive target material 11 can be combined with a charged non-magnetic target material 11. In this example, a magnetic field 70 can be applied in one direction to induce migration of the uncharged magnetically responsive target material 11 toward one of the opposing array surfaces 30, 30' or 32, 32' or 31, 31', and an electric field 62 can be applied in the opposite direction to induce migration of the charged non-magnetic target material toward the other of the opposing array surfaces 30, 30' or 32, 32' or 31, 31'. While several examples are provided, it is contemplated that other target material combinations and modalities can also be utilized.

[0234] Release of library fragments from the complex and sequencing With target material 11 immobilized on both opposing surfaces 30 and 30' or 32 and 32' or 31 and 31' of flow cell 24, flow cell 24 is ready for downstream analysis.

[0235] In embodiments utilizing complexes 10A, 10B immobilized on both opposing array surfaces 30 and 30' or 32 and 32', flow cell 24 is ready for release, amplification, and sequencing of library fragments.

[0236] An example of a method following immobilization and removal of unimmobilized target material (e.g., complexes 10A, 10B) includes initiating release of the sequenceable nucleic acid fragments 14, 14', 14" from the solid support 12 or 12' of the immobilized complexes 10A, 10B, thereby seeding at least some of the sequenceable nucleic acid fragments 14, 14', 14" onto primers 42, 42' of two opposing sequencing surfaces 30, 30' or 32, 32', respectively, and removing the solid support 12 or 12' and the unseeded sequenceable nucleic acid fragments 14, 14', 14" . These steps can be followed by any of the amplification techniques described herein, including those described with reference to Figures 9A-9C.

[0237] Prior to release of the fragments 14, 14', 14" an external fixative can be introduced into the flow cell 24. By way of example, the external fixative is air or a liquid or viscous medium that is immiscible with the target material 11 (specifically, complexes 10A, 10B) introduced into the flow cell 24. Air can also be used to aspirate wash fluid from the flow cell 24, creating droplets that surround the complexes 10A, 10B and form a diffusion barrier around each of the complexes 10A, 10B. The liquid or viscous external fixative at least partially surrounds the complexes 10A, 10B that are immobilized within the flow cell 24. The external fixative helps minimize diffusion of the sequenceable nucleic acid fragments 14, 14', 14" as they are released from the solid support 12 or 12'. If the external fixative is a temperature-responsive material, raising the temperature to the seeding temperature may cause the fixative to become more viscous and assume a form that can further minimize library diffusion.

[0238] Release of the sequenceable nucleic acid fragments 14, 14', 14" from the solid support 12 or 12' can then be initiated. In one example, a cleaving agent can be introduced into the flow cell 24, and a stimulus can be applied to trigger the cleaving agent to release the sequenceable nucleic acid fragments 14, 14', 14" from the solid support 12 or 12'. In another example, release of the sequenceable nucleic acid fragments 14, 14', 14" can include heating the flow cell 24 above the melting temperature of the primers hybridized to the fragments 14, 14', 14".

[0239] Upon release, transport and seeding of the sequenceable nucleic acid fragments 14, 14', or 14" can be restricted by an external fixation agent. Thus, the fragments 14, 14', or 14" of any particular complex 10A, 10B can be confined to a region of the sequencing surface 30, 30' or 32, 32' near the particular complex 10A, 10B from which the fragments 14, 14', or 14" were released.

[0240] The primers 42, 42' on the respective sequencing surfaces 30, 30' or 32, 32' of the flow cell 24 can seed the released sequenceable nucleic acid fragments 14, 14', or 14'. Seeding is achieved by hybridization between a first or second sequence of the fragments 14, 14', or 14" and a complementary sequence of the primers 42, 42' on the respective sequencing surfaces 30, 30' or 32, 32'. Seeding can be performed at a hybridization temperature appropriate for the fragments 14, 14', or 14" and the primers 42, 42'. In one example, seeding is performed at approximately 80°C, after which the temperature is reduced to room temperature (e.g., 25°C).

[0241] The location of seeding of the sequenceable nucleic acid fragments 14, 14', or 14" within the flow cell 24 depends, in part, on how the primers 42, 42' are attached. In the example of a flow cell 24 having an unpatterned sequencing surface 30, 30', the released sequenceable nucleic acid fragments 14, 14', or 14" are seeded across the polymer hydrogel 40, 40' in the recessed regions 38, 38'. In the example of a flow cell 24 having a patterned sequencing surface 32, 32', the released sequenceable nucleic acid fragments 14, 14', or 14" are seeded across the polymer hydrogel 40, 40' within each of the depressions 48, 48'.

[0242] An example of sequenceable nucleic acid fragments 14, 14', or 14'' seeded into different depressions 48, 48' along the patterned sequencing surface 32, 32' of flow cell 24 is shown in Figure 9A.

[0243] The solid supports 12, 12' may then be removed from the flow cell 24. Removal of the solid supports 12, 12' may involve any suitable technique, depending on the mechanism of attachment of the solid supports 12, 12' to the capture sites 44, 44'. By way of example, denaturation, bond cleavage, etc. may be used. Removal of the solid supports 12, 12' may also remove unseeded sequenceable nucleic acid fragments 14, 14', 14''. Removal of the solid supports 12, 12' may also remove external fixatives in liquid or viscous form.

[0244] The seeded sequencing library fragments 14, 14', 14'' can be amplified using cluster generation.

[0245] In one example of cluster generation, the sequenceable nucleic acid fragments 14, 14', or 14" are copied from the hybridized primers 42, 42' by 3' extension using a high-fidelity DNA polymerase. The high-fidelity DNA polymerase may be part of the amplification mixture introduced into the flow cell 24. The amplification mixture may also include other suitable polymerase chain reaction reagents. The original sequenceable nucleic acid fragments 14, 14', or 14" are denatured, and the copies remain immobilized on the sequencing surface 30, 30' or 32, 32'. Isothermal bridge amplification or some other form of amplification may be used to amplify the immobilized copies. For example, the copied template loops over and hybridizes to the adjacent complementary primer 42, 42', and the polymerase copies the copied template to form a double-stranded bridge structure that is denatured to form two single strands. These two strands loop over and hybridize to adjacent complementary primers 42, 42', where they are extended again to form two new double-stranded loops. This process is repeated for each template copy through cycles of isothermal denaturation and amplification, creating dense clonal clusters. Each cluster of double-stranded bridge structures is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. Clustering results in the formation of several template polynucleotide strands along the sequencing surface 30, 30' or 32, 32'. An example of this clustering is bridge amplification, which is one example of a possible amplification technique. It should be understood that other amplification techniques, such as the Exclusion Amplification (Examp) workflow (Illumina Inc.), can also be used.

[0246] Another example of amplification, and thus cluster generation, involves the use of a temperature-responsive material. This example is shown schematically in Figures 9A to 9C. This exemplary method includes introducing an amplification mixture including a temperature-responsive material in liquid form 63 into flow cell 24, gelling the temperature-responsive material in liquid form 63 (thereby producing a temperature-responsive material in gel form 63'), initiating amplification of seeded sequenceable nucleic acid fragments 14, 14', 14" to produce template strands 64, whereby the temperature-responsive material in gel form 63' reduces diffusion of template strands 64, liquefying the temperature-responsive material in gel form 63' (thereby producing a temperature-responsive material in liquid form 63), and removing the temperature-responsive material in liquid form 63 from flow cell 24.

[0247] 9A, an amplification mixture including a temperature-responsive material in liquid form 63 is introduced into flow channel 28, for example, via an inlet. In addition to the temperature-responsive material in liquid form 63, this example amplification mixture also includes a high-fidelity DNA polymerase and any other suitable polymerase chain reaction reagents.

[0248] The temperature-responsive material can transition from liquid form 63 to gel form 63' by changing the temperature conditions to which the material is exposed. In liquid form 63, the molecules of the temperature-responsive material are not linked and therefore can flow. In gel form 63', the molecules of the temperature-responsive material are cross-linked and therefore cannot flow. Gel form 63' contains pores, channels, or other openings that i) facilitate the diffusional exchange of small molecules, proteins, and reagents to access the seeded sequenceable nucleic acid fragments 14, 14', 14" for amplification, and ii) can impede or prevent the movement of the seeded sequenceable nucleic acid fragments 14, 14', 14" or template strands 64 by diffusion or convection. Thus, any temperature-sensitive material can i) facilitate in-gel amplification, ii) restrict diffusion, convection, or other movement of the seeded sequenceable nucleic acid fragments 14, 14', 14'' and template strands 64, iii) be pumped or flow as a liquid before crosslinking, iv) controllably crosslink and gel, and v) controllably delink and liquefy.

[0249] Examples of temperature-responsive materials include disulfide-crosslinked polyacrylamide, agarose, alginate, and copolymers of poly(N-isopropylacrylamide) (PNIPAAm) and polyethylene glycol (PEG). For each of these materials, amplification can be carried out at a temperature that does not melt the gel form 63'.

[0250] Copolymers of PNIPAAm and PEG are liquids at low temperatures and gels at high temperatures. An example of a copolymer of PNIPAAm and PEG is liquid at temperatures below 29°C and gels at temperatures above 32°C. The gelation temperature of the copolymer of PNIPAAm and PEG can be adjusted by varying the ratio of poly(N-isopropylacrylamide) to polyethylene glycol in the copolymer.

[0251] The amplification mixture is loaded into flow cell 24 under conditions in which the amplification reaction will not occur. For example, since amplification will not occur at 4° C., the amplification mixture (including the temperature-responsive material in liquid form 63) may be introduced at this temperature.

[0252] Inducing gelation of the temperature-responsive material in liquid form 63, thus producing gel form 63', can be accomplished by adjusting the temperature of flow cell 24 and the temperature-responsive material contained therein to the gelation temperature of the temperature-responsive material. Gel form 63' is shown in Figure 9B. The temperature to which flow cell 24 is adjusted will depend on the temperature-responsive material used.

[0253] As shown in FIG. 9B, amplification of the seeded sequenceable nucleic acid fragments 14, 14', 14" is initiated to produce template strands 64. Amplification can be initiated by adjusting the temperature of flow cell 24 and the amplification mixture contained therein to a temperature at which the PCR reagents are active. During amplification, the temperature-responsive material of gel form 63' reduces the migration of the seeded sequenceable nucleic acid fragments 14, 14', 14" and template strands 64.

[0254] Liquefying the temperature responsive material in gel form 63' to produce liquid form 63 can be performed by adjusting the temperature of flow cell 24 and the temperature responsive material contained therein back to the liquefaction temperature of the temperature responsive material. Again, the temperature to which flow cell 24 is adjusted will depend on the temperature responsive material used.

[0255] Liquid form 63 can then be pumped out of flow cell 24, and flow cell 24 can be prepared for subsequent sequencing. Flow cell 24 after the temperature-responsive material of liquid form 63 has been removed is shown in Figure 9C.

[0256] In one particular example, a copolymer of PNIPAAm and PEG is used in an amplification mixture in conjunction with recombinase-mediated polymerase chain reaction (PCR). Because typical recombinase-mediated isothermal PCR is inactive at 4°C and active at 37°C or other elevated temperatures, a temperature program can be used to control amplification; the copolymer of PNIPAAm and PEG is liquid at temperatures below 29°C and gels at temperatures above 32°C. In this example, the amplification mixture can be introduced into flow cell 24 as a liquid mixture at approximately 4°C. The temperature can then be raised to approximately 37°C to gel the copolymer and initiate PCR amplification. Upon completion, the copolymer in gel form 63' can be liquefied by lowering the temperature to below 29°C, e.g., approximately 8°C (which is a suitable sequencing temperature). The liquid form 63 can then be pumped out of flow cell 24, which can then be prepared for subsequent sequencing.

[0257] The use of temperature-responsive materials 63, 63′ can minimize diffusion of the seeded sequenceable nucleic acid fragments 14, 14′, or 14″ and the diffusion of amplified template strands 64 that migrate (e.g., as a result of diffusion or natural convection) to nearby depressions 48, 48′ of the patterned sequencing surface 32, 32′ or away from the initial seeding locations on the unpatterned sequencing surface 30, 30′. By limiting or preventing this migration, the clusters remain in relatively isolated regions of the flow cell 24, thereby allowing each of the clusters to be read individually without redundancy. Migration can also generate hybrid molecules not present in the original sequencing library fragments 14, 14′, 14″, which can result in inaccurate sequencing data. By limiting or preventing this migration, these hybrid molecules are not generated, improving the accuracy of the resulting sequencing data.

[0258] Although Figures 9A-9C show a flow cell 24 having a patterned sequencing surface 32, 32', it should be understood that the method can also be performed using an unpatterned sequencing surface 30, 30'.

[0259] Furthermore, the methods illustrated in Figures 9A-9C can be performed with any sequenceable nucleic acid fragments 14, 14', 14" including those not tethered to a solid support 12, 12'. In this example, any suitable library preparation technique that adds desired adapters to a fragmented DNA sample can be used. The sequenceable nucleic acid fragments 14, 14', 14" can be introduced and seeded onto the sequencing surface 30, 30' or 32, 32 of a flow cell. Once the library fragments are seeded, the methods described in Figures 9A-9C can be performed.

[0260] It should further be understood that the methods shown in Figures 9A to 9C may not be performed on clustered solid supports 13, as these target materials 11 are not exposed to amplification on flow cells 24.

[0261] Next, a sequencing primer can be introduced that hybridizes to a complementary sequence on the template polynucleotide strand, preparing the template polynucleotide strand 64 for sequencing. The 3' end of the template 64 and the flow cell-bound primer 42, 42' (not attached to the copy) can be blocked to prevent interference with the sequencing reaction, particularly to prevent undesired priming.

[0262] To initiate sequencing, an incorporation mixture can be added to flow cell 24. In one example, the incorporation mixture includes a liquid carrier, a polymerase, and fluorescently labeled nucleotides. The fluorescently labeled nucleotides can include a 3'OH blocking group. When the incorporation mixture is introduced into flow cell 24, the fluid enters flow channel 28 and, in some examples, into recess 48, 48' (where the template polynucleotide strand resides).

[0263] Fluorescently labeled nucleotides are added to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner, and detection of the order and type of nucleotides added to the sequencing primer can be used to sequence the template. More specifically, one of the nucleotides is incorporated by each polymerase into a nascent strand that extends the sequencing primer and is complementary to the template polynucleotide strand. In other words, for at least some of the template polynucleotide strands spanning flow cell 24, each polymerase extends a sequencing primer hybridized with one of the nucleotides in the incorporation mixture.

[0264] Nucleotide incorporation can be detected through an imaging event during which an illumination system (not shown) can provide excitation light to the respective sequencing surfaces 30, 30' or 32, 32'.

[0265] In some instances, the nucleotide may further comprise a reversible termination feature (e.g., a 3'OH blocking group) that terminates further primer extension once the nucleotide is added to the sequencing primer. For example, a nucleotide analog having a reversible terminal moiety may be added to the sequencing primer such that further extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, in instances using a reversible terminus, a deblocking reagent may be delivered to flow cell 24 after detection has occurred.

[0266] Wash(es) may be performed between the various fluid delivery steps. The SBS cycle is then repeated n times to extend the sequencing primer by n nucleotides, thereby allowing detection of a sequence of length n.

[0267] In some instances, the forward strand may be sequenced and removed, and then the reverse strand may be constructed and sequenced as described herein.

[0268] While SBS is described in detail, it should be understood that the flow cell 24 described herein can be utilized with other sequencing protocols, for genotyping, or in other chemical and / or biological applications. In some cases, the primers 42, 42' of the flow cell 24 may be selected to enable simultaneous paired-end sequencing, where both the forward and reverse strands are present on the polymer hydrogel 40, 40', allowing simultaneous base calling of each read. Sequential and simultaneous paired-end sequencing facilitates the detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.

[0269] Clustered solid supports and sequencing As described above, with target material 11 immobilized on both opposing surfaces 30 and 30', or 32 and 32', or 31 and 31' of flow cell 24, flow cell 24 is ready for downstream analysis. When clustered solid supports 13 are immobilized on both opposing surfaces 31 and 31' of flow cell 24, flow cell 24 is ready for sequencing. In these instances, amplification and cluster generation occurs on solid supports 12 or 12', away from flow cell 24, and flow cell 24 is ready for sequencing.

[0270] Sequencing can be performed as described herein by introducing a sequencing primer and an incorporation mixture and performing sequential sequencing cycles.

[0271] To further illustrate the present disclosure, examples are presented herein. It should be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure.

[0272] Non-limiting working examples Example 1 Complexes similar to those shown in Figure 1A were prepared with an average diameter of 3 μm. The solid support for the complexes was DYNABEAD™ M-280 streptavidin beads from ThermoFisherScientific. The solid supports were approximately 1.18 g / cm2 each.3 The library fragments were attached to the solid support via desthiobiotin oligos, which have a weaker affinity for streptavidin on the bead surface than biotin. The library fragments contained P5' and P7 sequences along with the index sequence, and included read 1 and read 2 sequences.

[0273] The complexes were loaded onto a flow cell containing opposing patterned sequencing surfaces (containing P5 and P7 primers) using an example method similar to that described in Figures 3A and 3B.

[0274] More specifically, the composite is first divided into two fluids, the first of which is approximately 2 g / cm 3 and the second fluid has a density of about 1 g / cm 3 The first fluid was a 1 g / ml sodium polytungstate solution (500 mg sodium polytungstate per 500 μL of sodium citrate saline buffer containing sodium dodecyl sulfate) containing the conjugate at a concentration of 600,000 particles per μL. The second fluid was a saline sodium citrate buffer containing sodium dodecyl sulfate containing the conjugate at a concentration of 600,000 particles per μL.

[0275] The first fluid was introduced into the flow cell, causing the complex to be immobilized on the top surface of the flow cell. The flow cell was then washed with a wash solution. The second fluid was introduced into the flow cell, causing the complex to be immobilized on the bottom surface of the flow cell. Attachment of the complex to each surface was achieved using an anchor (e.g., a complementary primer containing biotin hybridized to a P5 primer bound to the gel material, or click chemistry was used to covalently attach an alkyne-PEG-biotin linker to a free azide on the gel material).

[0276] Figure 10A shows a bright-field image of the top surface after immobilization of the complex, and Figure 10B shows a bright-field image of the bottom surface after immobilization of the complex. The dark areas in each image represent the immobilized complex.

[0277] Free biotin in a saline-sodium citrate buffer containing sodium dodecyl sulfate was introduced, and the flow cell was heated to approximately 80°C to release the library from each complex. Clustering was performed using isothermal amplification. The clusters were stained with Sytox green, and the resulting images (not reproduced here) confirm the formation of clusters of template strands on each sequencing surface of the flow cell.

[0278] Sequencing was then performed on the flow cell, and portions of the sequencing data collected on the top and bottom surfaces of the flow cell are shown in Figures 11A and 11B.

[0279] Figure 11A shows a histogram of molecular coverage on the top and bottom of one lane of a flow cell. This data indicates the extent and uniformity of sequencing coverage of the lane.

[0280] Figure 11B shows the percentage of Q-scores greater than Q30 for various sequencing cycles on the top and bottom of one lane of a flow cell. A Q-score of 30 (Q30) corresponds to a 1 in 1000 probability of an incorrect base call. This means that the base-call accuracy (i.e., the probability of a correct base call) is 99.9%. A base-call accuracy as low as 99% (Q20) has a 1 in 100 probability of an incorrect base call, meaning that every 100 base pair sequencing read contains an error. When sequencing quality reaches Q30, virtually all reads are perfect and free of errors and ambiguities. As shown in Figure 11B, the percentage of Q-scores greater than Q30 generally ranges from 60% to 99% across all sequencing cycles.

[0281] All data collected confirmed that the denser fluid (in this example, fluid 1) was compatible with the sequencing surface of the flow cell.

[0282] Example 2 Complexes similar to those shown in Figure 1A were prepared with an average diameter of 3 μm. The solid support for the complexes was DYNABEAD™ M-280 streptavidin beads from ThermoFisherScientific. The solid supports were approximately 1.18 g / cm2 each. 3 The fragments on a particular bead were from the same long DNA molecule (from the PhiX genome).

[0283] In this example, flow cell lanes (opposing surfaces coated with gel material) were prepared with varying concentrations of capture moieties (i.e., alkyne-PEG-biotin linkers). These linkers were covalently attached to free azides on the gel material of the flow cell lanes using click chemistry. The flow cell lanes were washed and exposed to alkyne-PEG-biotin solutions at concentrations of about 0.5 μM, about 5 μM, or about 25 μM, respectively. The solutions were allowed to incubate at about 60° C. for about 30 minutes. The flow cell lanes were then washed again.

[0284] The composite is first divided into two fluids, the first fluid being approximately 1 g / cm 3 and the second fluid has a density of about 2 g / cm 3 The first fluid was a saline sodium citrate buffer containing sodium chloride, containing the conjugate at a concentration of 25,000 per μL. The second fluid was a 2 g / ml sodium polytungstate solution, containing the conjugate at a concentration of 25,000 per μL.

[0285] The first fluid was introduced into each flow cell lane, thereby immobilizing the complex on the bottom surface of the flow cell lane. The aspiration rate was 100 μL / min, and the first fluid was allowed to dwell in the flow cell for 180 seconds. The flow cell was then washed with a wash solution. The second fluid was introduced into each flow cell lane, thereby immobilizing the complex on the top surface of the flow cell lane. The aspiration rate was 100 μL / min, and the second fluid was allowed to dwell in the flow cell lane for 450 seconds. The flow cell lane was then washed with a wash solution.

[0286] The bottom and top surfaces of each flow cell lane were imaged, and the microscopic images were used to count the complexes (beads) immobilized on each surface.

[0287] 1mm on the bottom 2 The number of beads per mm is shown in Figure 12A. 2 The number of beads per mm is shown in Figure 12B. The concentration of each bar in Figures 12A and 12B represents the alkyne-PEG-biotin concentration (about 0.5 μM, about 5 μM, or about 25 μM) used to prepare the flow cell prior to immobilization of the complex. As shown, the alkyne-PEG-biotin concentrations were such that each of these yielded about 2,100 beads / mm. 2 to approximately 2,300 beads / mm 2 The number of complexes immobilized on the top surface was approximately 550 beads / mm 2 Approximately 1,150 beads / mm 2 On the top side, the lane treated with a high concentration of alkyne-PEG-biotin linker had a higher number of immobilized complexes / beads on that lane.

[0288] These results indicate that heavier fluids help immobilize the complexes on the top surface, and that increasing the concentration of capture sizes on the top surface may also aid in immobilization.

[0289] Example 3 Complexes similar to those shown in Figure 1A were prepared with an average diameter of 3 μm. The solid support for the complexes was DYNABEAD™ M-280 streptavidin beads from ThermoFisherScientific. The solid supports were approximately 1.18 g / cm2 each. 3 The fragments on a particular bead were from the same long DNA molecule (from the PhiX genome).

[0290] In this example, eight flow cell lanes (opposing surfaces coated with gel material) were prepared with capture moieties (i.e., alkyne-PEG-biotin linkers). These linkers were covalently attached to free azides on the gel material of the flow cell lanes using click chemistry. The flow cell lanes were washed and each exposed to an alkyne-PEG-biotin solution at a concentration of approximately 5 μM. The solution was incubated at approximately 60°C for approximately 30 minutes. The flow cell lanes were then washed again.

[0291] The composite is first divided into two fluids, the first fluid being approximately 1 g / cm 3 and the second fluid has a density of about 2 g / cm 3 The first fluid was a sodium citrate buffer solution containing the conjugate at a concentration of 40,000 per μL. The second fluid was a 2 g / ml sodium polytungstate solution containing the conjugate at a concentration of 40,000 per μL.

[0292] The first fluid was introduced into seven of the flow cell lanes, thereby immobilizing the complexes on the bottom surface. The aspiration rate was 100 μL / min, and the first fluid was allowed to dwell in each lane for 240 seconds. The flow cell lanes were then washed with a wash solution. The second fluid was introduced into each of the seven flow cell lanes, thereby immobilizing the complexes on the top surface. The aspiration rate ranged from 80 μL / ms to 100 μL / ms, and the second fluid was allowed to dwell in the flow cell for 300 seconds. The flow cell lanes were then washed with a wash solution.

[0293] In lane 8, the fluids were diluted to 100 μL each and each fluid injection was performed twice, so lane 8 had a double loading.

[0294] The bottom and top surfaces of each flow cell lane were imaged, and the immobilized complexes (beads) on each surface were counted. Table 1 shows the 1 mm 2 The average number of beads per sample is shown. [Table 1]

[0295] The target number of complexes (beads) on each surface is 4,000 beads / mm 2 Lanes 1-7 are slightly below target, but the number of complexes on the top and bottom surfaces of these lanes is relatively consistent. Lane 8 (exposed to double loading) exceeds the target number of complexes on both surfaces.

[0296] Figure 13A shows the target bead count measured along the length of flow cell lane 1 from inlet (1) to outlet (5) and the number of beads per mm 2 Figure 13B shows the target number of beads and the number of beads per mm measured along the length of flow cell lane 7 from inlet (1) to outlet (5). 2 The numbers show the number of beads per 1000 μm. Measurements were taken at equal distances along the length. These results show that immobilization is relatively consistent along the length of the lanes in both the top and bottom flow channels.

[0297] Example 4 Complexes similar to those shown in Figure 1A were prepared with an average diameter of 3 μm. The solid support for the complexes was DYNABEAD™ M-280 streptavidin beads from ThermoFisherScientific. The solid supports were approximately 1.18 g / cm2 each. 3 The fragments on a particular bead were from the same long DNA molecule (from the PhiX genome).

[0298] In this example, 10 flow cell lanes (opposing surfaces coated with gel material) were prepared with capture moieties (i.e., alkyne-PEG-biotin linkers). These linkers were covalently attached to free azides on the gel material of the flow cell lanes using click chemistry. The flow cell lanes were washed and then each exposed to an alkyne-PEG-biotin solution at a concentration of approximately 5 μM. The solution was incubated at approximately 60°C for approximately 30 minutes. The flow cell lanes were then washed again.

[0299] The composite is first divided into two fluids, the first fluid being approximately 1 g / cm 3 and the second fluid has a density of about 2 g / cm 3 The first fluid was a sodium citrate buffer solution containing 10 μg of conjugate per 50 μL, and the second fluid was a 2 g / ml sodium polytungstate solution containing 12.5 μg of conjugate per 50 μL.

[0300] The first fluid was introduced into 10 flow cell lanes, and the complexes were immobilized on the bottom surface. The aspiration rate was 100 μL / min, and the first fluid was allowed to dwell in each lane for 300 seconds. The flow cell lanes were then washed with a wash solution. The second fluid was introduced into each of the 10 flow cell lanes, and the complexes were immobilized on the top surface. The aspiration rate was 80 μL / ms, and the second fluid was allowed to dwell in the flow cell for 360 seconds. The flow cell lanes were then washed with a wash solution.

[0301] The bottom and top surfaces of each flow cell lane were imaged, and the complexes (beads) immobilized on each surface were counted.

[0302] Figure 14 shows the target number of beads and the distance measured along the length of one lane of the flow cell from the inlet (1) to the outlet (10) in 1 mm 2 Figure 14 shows the number of beads per 100 μm. Figure 14 also shows a linear fit of the top and bottom data. These results demonstrate that when complexes are introduced according to the example methods disclosed herein, immobilization is relatively consistent along the length of the top and bottom surfaces of the flow channel.

[0303] Example 5 Complexes similar to those shown in Figure 1A were prepared with an average diameter of 3 μm. The solid support for the complexes was DYNABEAD™ M-280 streptavidin beads from ThermoFisherScientific. The solid supports were approximately 1.18 g / cm2 each. 3 The library fragments on a particular bead were from the same long DNA molecule (from the PhiX genome). The library fragments were attached to the solid support via desthiobiotin oligos, which have a weaker affinity for streptavidin on the bead surface than biotin.

[0304] In this example, eight flow cell lanes (opposing surfaces coated with gel material) were prepared with capture moieties (i.e., alkyne-PEG-biotin linkers). These linkers were covalently attached to free azides on the gel material of the flow cell lanes using click chemistry. The flow cell lanes were washed and each exposed to an alkyne-PEG-biotin solution at a concentration of approximately 5 μM. The solution was incubated at approximately 60°C for approximately 30 minutes. The flow cell lanes were then washed again.

[0305] The composite is first divided into two fluids, the first fluid being approximately 1 g / cm 3 and the second fluid has a density of about 2 g / cm 3 The first fluid was a sodium citrate buffer solution containing 10 μg of conjugate per 50 μL, and the second fluid was a 2 g / ml sodium polytungstate solution containing 12.5 μg of conjugate per 50 μL.

[0306] The first fluid was introduced into eight of the flow cell lanes, thereby immobilizing the complexes on the bottom surface. The aspiration rate was 100 μL / min, and the first fluid was allowed to dwell in each lane for 240 seconds. The flow cell lanes were then washed with a wash solution. The second fluid was introduced into each of the eight flow cell lanes, thereby immobilizing the complexes on the top surface. The aspiration rate ranged from 80 μL / ms to 100 μL / ms, and the second fluid was allowed to dwell in the flow cell for 300 seconds. The flow cell lanes were then washed with a wash solution.

[0307] The bottom and top surfaces of each flow cell lane were imaged, and the complexes (beads) immobilized on each surface were counted.

[0308] Free biotin in sodium citrate buffer was introduced, and the flow cell was heated to approximately 80°C to release the library from each complex. Clustering was performed using bridge amplification. Sequencing was then performed on the flow cell. The collected sequencing data included a passing filter (%PF) (percentage). Passing filter (PF) is a metric used to describe clusters that pass a chastity threshold and are used for further processing and analysis of the sequencing data. A higher passing filter (%) result indicates an increased yield of unique clusters used in the sequencing data.

[0309] Table 2 shows the 1 mm 2 The average number of beads per well and PF data for each lane are shown. [Table 2]

[0310] The target number of complexes (beads) on each surface of lanes 1–7 is 4,000 beads / mm 2 (Total 8,000 beads / mm 2 The target number of complexes (beads) on each surface in lane 8 was 5,500 beads / mm 2 (Total 11,000 beads / mm 2) Lanes 1–8 are slightly below target, but the total number of complexes at the top and bottom of these lanes is relatively consistent. The passing filter data indicate that the majority of the nanowells were occupied by monoclonal clusters.

[0311] Additional Notes Furthermore, ranges provided herein should be understood to include the stated range and any value or subrange within the stated range, as if expressly recited. For example, a range expressed by about 2 mm to about 300 mm should be interpreted to include not only the explicitly stated 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 subranges such as about 20 mm to about 225 mm.

[0312] It is understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein. Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein. It is also understood that terms used expressly herein, and which may also appear in any disclosures incorporated by reference, are to be given the meaning most consistent with the particular concepts disclosed herein.

[0313] Although several embodiments have been described in detail, it should be understood that the disclosed examples may be modified, and therefore the foregoing description should be considered non-limiting.

Claims

1. A method comprising the steps of immobilizing a plurality of target materials on each of two opposing sequencing surfaces of a flow cell, the target material is either i) a complex comprising a solid support and a sequenceable nucleic acid fragment attached to the solid support, or ii) a clustered solid support comprising a solid support and a cluster of template strands attached to the solid support; Immobilization is introducing a first fluid having a first portion of the plurality of target materials therein into the flow cell, thereby immobilizing at least a portion of the target materials by capture sites on one of the two opposing sequencing surfaces; removing the first fluid and any unimmobilized target material from the flow cell; introducing a second fluid having a second portion of the plurality of target materials therein into the flow cell, thereby immobilizing at least a portion of the target materials by capture sites on the other one of the two opposing sequencing surfaces; Including, where: the first fluid has a density lower than that of the target material and the second fluid has a density higher than that of the target material; or the second fluid has a density lower than that of the target material and the first fluid has a density higher than that of the target material; and The method, wherein the second portion of the target material is not identical to the first portion of the target material.

2. 2. The method of claim 1, wherein the first or second fluid having a density lower than that of the target material is a buffered aqueous solution, and the second or first fluid having a density higher than that of the target material is a sodium polytungstate solution or a sodium chloride solution.

3. The density of the first or second fluid at the capture temperature is at least 0.1 g / cm greater than the density of the target material at the capture temperature. ３ and the density of the second or first fluid at the capture temperature is at least 0.1 g / cm lower than the density of the target material at the capture temperature. ３ The method of claim 1 or 2, wherein the concentration is high.

4. The density of the first or second fluid is less than the density of the target material and is about 1 g / cm at the capture temperature. ３ and the density of the second or first fluid is greater than the density of the target material by about 2 g / cm at the capture temperature. ３ 3. The method according to claim 1, wherein

5. The method of any one of claims 1 to 4, further comprising the step of allowing a predetermined time to elapse before removing the first fluid and any unimmobilized target material from the flow cell.

6. 6. The method of claim 1, wherein the target material immobilized on one of the two opposing sequencing surfaces remains immobilized on one of the two opposing sequencing surfaces when the second fluid is introduced.

7. The target material is a solid support; a sequenceable nucleic acid fragment attached to said solid support; The method according to any one of claims 1 to 6, wherein the complex comprises:

8. Removing the second fluid and any unimmobilized complexes from the flow cell; initiating release of the sequenceable nucleic acid fragments from the solid support of the complexes immobilized in the flow cell by heating the flow cell above the melting temperature of the primers, thereby seeding at least some of the sequenceable nucleic acid fragments onto the primers on each of the two opposing sequencing surfaces; removing the solid support and unseeded sequenceable nucleic acid fragments; introducing an amplification mixture into the flow cell, the amplification mixture including a temperature responsive material in liquid form; allowing the temperature responsive material in the liquid form to gel; initiating amplification of the seeded sequenceable nucleic acid fragments to generate template strands, whereby the temperature-responsive material in gel form reduces diffusion of the template strands; liquefying the temperature-responsive material in the gel form; removing the liquid form of the temperature responsive material from the flow cell; The method of claim 7 further comprising:

9. The method of claim 8, wherein the temperature-responsive material is a copolymer of poly(N-isopropylacrylamide) and polyethylene glycol.

10. The target material: a solid support; a cluster of template strands attached to the solid support; 2. The method of claim 1, wherein the clustered solid support comprises:

11. A preparation fluid having a target material therein; a first introduction fluid having a density lower than the density of the target material; a second introduction fluid having a density greater than the density of the target material; 11. A kit for use in the method of any one of claims 1 to 10, comprising:

12. 12. The kit of claim 11, wherein the first induction fluid is a buffered aqueous solution and the second induction fluid is a sodium polytungstate solution or a sodium chloride solution.

13. 13. The kit of claim 12, wherein the second induction fluid is the sodium polytungstate solution, the sodium polytungstate solution having a concentration of about 1 gram of sodium polytungstate per milliliter of water.

14. The density of the first input fluid at the capture temperature is at least 0.1 g / cm greater than the density of the target material at the capture temperature. ３ and the density of the second input fluid at the capture temperature is at least 0.1 g / cm lower than the density of the target material at the capture temperature. ３ The kit according to any one of claims 11 to 13,

15. The density of the first inlet fluid is about 1 g / cm at the capture temperature. ３ and the density of the second input fluid is about 2 g / cm at the capture temperature. ３ The kit according to claim 11 or 14,

16. The kit of any one of claims 11 to 15, further comprising a flow cell having two opposing sequencing surfaces.

17. Each of the opposing sequencing surfaces comprises: a polymer hydrogel; amplification primers attached to the polymer hydrogel; Chemical capture sites and 17. The kit of claim 16, comprising:

18. The chemical capture moiety is one member of a binding pair, 18. The kit of claim 17, wherein the target material is a solid support coated with the other member of the binding pair.

19. The target material: a solid support; a sequenceable nucleic acid fragment attached to said solid support; The kit according to any one of claims 11 to 18, which is a complex comprising:

20. The target material: a solid support; a cluster of template strands attached to the solid support; The kit of any one of claims 11 to 18, wherein the solid support is a clustered solid support comprising:

21. The kit of any one of claims 11 to 19, further comprising an amplification mixture comprising a temperature-responsive material in liquid form.

Images