Biotin-Streptavidin Cleavage Composition and Library Fragment Cleavage

Abstract

The problem that the present invention seeks to solve is to provide compositions and methods that can be used to cleave library fragments from the solid support to which they are attached. [Solution] One example of a biotin-streptavidin cleavage composition provided by the present invention includes a formamide reagent and a salt buffer. The formamide reagent is present in the biotin-streptavidin cleavage composition in an amount ranging from about 10% to about 50%, based on the total volume of the biotin-streptavidin cleavage composition. The salt buffer constitutes the remainder of the biotin-streptavidin cleavage composition. In some examples, the biotin-streptavidin cleavage composition is used to cleave library fragments from a solid support. In other examples, other mechanisms are used to cleave the library fragments from the solid support.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 969,440, filed February 3, 2020, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Biological arrays are one of a wide range of tools used to detect, analyze, and / or purify molecules, including proteins, nucleic acids, and the like. In some applications, arrays are engineered to include probes capable of capturing molecules of interest. In other applications, arrays are engineered with one member of a binding pair, and the molecules of interest are labeled or tagged with the other member of the binding pair. Examples of binding pairs include biotin and avidin or streptavidin. The affinity of avidin or streptavidin for biotin is one of the strongest non-covalent biological interactions. Furthermore, biotin labels rarely interfere with the function of the labeled molecule. These properties make biotin and avidin or biotin and streptavidin particularly desirable for a variety of biological applications.

[0003] In some biological applications, it is desirable to tether or link a molecule of interest in a conditional and controlled manner, cleaving the tether or link at a specific time point and controlling the release of the cleaved molecule of interest. In some cases, the strength of the biotin-avidin or biotin-streptavidin interaction can make conditional and / or controlled cleavage difficult. Summary of the Invention

[0004] Disclosed herein are compositions and methods that can be used to cleave library fragments from the solid support to which they are attached. One example of a composition can rapidly and efficiently cleave biotin-streptavidin bonds. Another example of a composition is activated under conditions that allow spatial release of the library fragments. One example of a method involves a two-step release of the library fragments from the solid support. The cleavage mechanisms of the two-step release are orthogonal; therefore, the first cleavage mechanism is unstable and the second cleavage mechanism is stable. The second cleavage mechanism allows spatial release of the library fragments.

[0005] The compositions and methods may be used on flow cells. Each of the embodiments disclosed herein can be used on a flow cell surface without adversely affecting the chemistry on the flow cell surface (e.g., polymeric hydrogels, amplification primers, etc.).

[0006] A first aspect disclosed herein is a biotin-streptavidin cleavage composition, which comprises about 10% to about 50% by volume of a formamide reagent, with the remainder being a salt buffer.

[0007] A second aspect disclosed herein is a biotin-streptavidin cleavage composition, the biotin-streptavidin cleavage composition consisting of about 10% to about 50% by volume of a formamide reagent comprising formamide and an optional buffer, with the remainder being a salt buffer comprising sodium chloride, sodium citrate, and a biocompatible surfactant.

[0008] A third aspect disclosed herein is a method, the method comprising: introducing library fragments into a flow cell, where the library fragments are attached to a streptavidin-coated solid support; introducing a biotin-streptavidin cleavage composition into the flow cell, where the biotin-streptavidin cleavage composition comprises about 10% to about 50% by volume of formamide reagent, with the remainder being a salt buffer; and allowing the biotin-streptavidin cleavage composition to incubate in the flow cell at a temperature ranging from about 60°C to about 70°C, thereby releasing at least some of the library fragments from the solid support and seeding them onto amplification primers on the surface of the flow cell.

[0009] A fourth aspect disclosed herein is a kit comprising: a streptavidin-coated solid support; an adapter sequence having biotin attached to one end thereof, such that the biotin becomes attached to the streptavidin-coated solid support; a sample fluid comprising a genomic sequence that is fragmented and becomes attached to the adapter sequence; and a biotin-streptavidin cleavage composition comprising about 10% to about 50% by volume of formamide reagent, with the remainder being a salt buffer.

[0010] A fifth aspect disclosed herein is a method, the method comprising: introducing desthiobiotinylated library fragments into a flow cell, where the desthiobiotinylated library fragments are attached to a streptavidin-coated solid support; introducing a cleavage composition into the flow cell, where the cleavage composition is at a temperature in the range of about 18°C ​​to about 30°C, and where the cleavage composition comprises free biotin and a salt buffer; and raising the temperature of the cleavage composition to about 60°C to about 70°C, thereby releasing at least some of the library fragments from the solid support and seeding them onto amplification primers on the surface of the flow cell.

[0011] A sixth aspect disclosed herein is a library preparation fluid, the library preparation fluid comprising a liquid carrier and library preparation beads in the liquid carrier, each library preparation bead comprising a solid support and a transposome complex attached to the solid support, the transposome complex comprising a transposase enzyme; a double-stranded molecule bound to the transposase enzyme, the double-stranded molecule comprising a transferred strand comprising a 3' transposon end sequence, an adapter sequence, a cleavage site, and a 5' linked end sequence, wherein the transferred strand comprises a 3' transposon end sequence, an adapter sequence, a cleavage site, and a 5' linked end sequence, wherein the adapter sequence and the 5' linked end sequence are adjacent to the cleavage site, and a non-transferred strand comprising the 3' transposon end sequence; and a splint sequence that hybridizes to at least a portion of the adapter sequence and at least a portion of the 5' linked end sequence so as to splint the cleavage site.

[0012] A seventh aspect disclosed herein is a method, the method comprising introducing a plurality of prepared library preparation beads into a reaction vessel, each of the prepared library preparation beads comprising a solid support and a plurality of bridge molecules attached to the solid support, each of the bridge molecules comprising a double-stranded DNA fragment and a transferred strand, the transferred strand being attached at its 5' end to each strand of the double-stranded DNA fragment, each transferred strand comprising a 3' transposon end sequence, a first adaptor sequence, a cleavage site, and a 5' ligation end sequence, wherein the first adaptor sequence and the 5' ligation end sequence are adjacent to the cleavage site, and a second adaptor sequence. introducing a second adapter sequence, the second adapter sequence being attached to each strand of the double-stranded DNA fragment at its 3' end, and a splint sequence 68 hybridized to at least a portion of the first adapter sequence and at least a portion of the 5' linked end sequence so as to splint the cleavage site; exposing the prepared library preparation beads to a cleavage agent to remove the cleavage site, thereby leaving a plurality of bridge molecules attached to the solid support via the splint; and heating the reaction vessel to a temperature at which the splint and double-stranded DNA fragments dissociate.

[0013] It should be understood that any feature of any one of these aspects may be combined together in any desired manner. Furthermore, it should be understood that any combination of the features of the first aspect, and / or the second aspect, and / or the third aspect, and / or the fourth aspect, and / or the fifth aspect, and / or the sixth aspect, and / or the seventh aspect may be combined with any of the embodiments disclosed herein to achieve the benefits described in this disclosure, including, for example, improved biotin-streptavidin bond cleavage.

[0014] 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]

[0015] [Figure 1A] 1A-1D are schematic diagrams of different examples of target materials disclosed herein. [Figure 1B] 1A-1D are schematic diagrams of different examples of target materials disclosed herein. [Figure 2A] 2A-2C are schematic diagrams of a portion of a library preparation bead before tagging of the non-transferred strand (2A), during tagging (2B), and after tagging and during ligation (2C). [Figure 2B] 2A-2C are schematic diagrams of a portion of a library preparation bead before tagging of the non-transferred strand (2A), during tagging (2B), and after tagging and during ligation (2C). [Figure 2C] 2A-2C are schematic diagrams of a portion of a library preparation bead before tagging of the non-transferred strand (2A), during tagging (2B), and after tagging and during ligation (2C). [Figure 3A] FIG. 2 is a top view of an example of a flow cell. [Figure 3B]FIG. 3B is an enlarged cross-sectional view of an example of a flow channel and unpatterned sequencing surface taken along line 3B-3B of FIG. 3A. [Figure 3C] 3C is an enlarged cross-sectional view of an example of a flow channel and patterned sequencing surface taken along line 3C-3C of FIG. 3A. [Figure 4] FIG. 1 is a schematic flow diagram showing the dual mechanism release of sequenceable library fragments from prepared library preparation beads. [Figure 5] 1 is a graph showing the fluorescence intensity of tetrachloro-fluorescein (TET) QC primer (Y-axis) versus the volume percent of formamide reagent in the cleavage composition (X-axis). [Figure 6] 1 is a bar graph showing the percentage of beads remaining after various washing steps. [Figure 7] 1 is a bar graph showing the percentage of desthiobiotinylated DNA release at different temperatures when exposed to free biotin or desthiobiotin under high salt conditions. DETAILED DESCRIPTION OF THE INVENTION

[0016] In some sequencing applications, DNA library fragments are introduced into a flow cell on a solid support (e.g., beads). The use of a solid support may be desirable because it can preserve the contiguity information of the longer genetic material from which the library fragments are generated. In some cases, DNA library fragments are attached to the solid support using biotin-streptavidin interactions. For library seeding and amplification on the flow cell, the biotinylated DNA library fragments must be released from the solid support. The inventors have found that some reagents strong enough to disrupt the biotin-streptavidin interaction also interfere with or otherwise adversely affect the hybridization of the biotinylated DNA library fragments to primers on the flow cell surface. As an example, strong denaturing reagents (e.g., about 95% formamide and about 10 mM ethylenediaminetetraacetic acid (EDTA)) and high temperature incubation (e.g., 90°C for 2 minutes) were found to disrupt the biotin-streptavidin interaction, but the released biotinylated DNA library fragments did not hybridize to the flow cell surface.

[0017] In one example, a cleavage composition is disclosed herein that can efficiently disrupt biotin-streptavidin bonds, completely releasing biotinylated DNA library fragments. Upon release, the biotinylated DNA library fragments can undergo hybridization in the cleavage composition. Thus, the cleavage composition disclosed herein efficiently disrupts biotin-streptavidin interactions, ensuring immediate hybridization. The cleavage composition is effective at temperatures below 70°C. 70°C is below the operating temperature of most flow cells, and therefore, use of the cleavage composition in a flow cell does not adversely affect the chemistry on the flow cell surface (e.g., polymer hydrogels, amplification primers, etc.).

[0018] The cleavage compositions disclosed herein can disrupt biotin-streptavidin bonds very quickly and effectively. In some cases, incubation times range from about 2 minutes to about 5 minutes. These times are significantly shorter than other methods, such as those involving sodium dodecyl sulfate (SDS), urea, and biotin, which may involve 15 minutes of shaking and 15 minutes of incubation.

[0019] The cleavage compositions disclosed herein may be used in other applications where it is desirable to disrupt the biotin-streptavidin binding pair.

[0020] In another example, biotinylated DNA library fragments are replaced with desthiobiotinylated DNA library fragments. In this example, cleavage is activated under conditions that allow spatial release of the library fragments. "Spatial release" means that the DNA fragments are released in the absence of inertial fluid mixing, and thus can controllably diffuse and seed onto the flow cell surface in proximity to the solid support from which the fragments are released. When cleavage occurs on the flow cell surface, the cleavage conditions do not adversely affect the chemistry on the flow cell surface (e.g., polymer hydrogels, amplification primers, etc.).

[0021] In yet another example, DNA library fragments are attached to a solid support with a dual release mechanism. The dual release mechanism includes a cleavage site and a splint, both of which retain the library fragments on the solid support, each of which involves a different release process. When library fragment release is desired, the cleavage site may be first removed using a suitable cleavage agent, and then the splint may be removed using heat. This example allows for controlled release of the library fragments because the cleavage conditions involve two orthogonal processes (one process does not initiate, affect, or otherwise interfere with the other).

[0022] definition

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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. For example, the terms can refer to ±10% or less from the stated value, e.g., ±5% or less from the stated value, e.g., ±2% or less from the stated value, e.g., ±1% or less from the stated value, e.g., ±0.5% or less from the stated value, e.g., ±0.2% or less from the stated value, e.g., ±0.1% or less from the stated value, e.g., ±0.05% or less from the stated value.

[0027] 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.

[0028] 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 contain any combination of nucleotides and / or nucleic acids. In some examples, adapters can contain a sequence complementary to at least a portion of a primer, such as a primer containing a universal nucleotide sequence (e.g., a P5 or P7 sequence). As a specific example, an adapter at one end of the sequence contains a sequence complementary to at least a portion of a first flow cell primer, and an adapter at the other end of the fragment contains a sequence identical to at least a portion of a second flow cell primer. The complementary adapter can hybridize to the first flow cell primer, and the identical adapter is a template for its complementary copy, which can hybridize to the second flow cell primer during clustering. In some examples, the adapter can contain 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.

[0029] Chemical capture site: A portion of a flow cell surface that is modified with chemical properties that allow for the localization of target materials (e.g., complexes, protein biomarkers, etc.). In one example, a capture site may 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)). An example of a chemical capture agent includes 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). Yet another example of a chemical capture agent is a chemical reagent that can form an electrostatic interaction, hydrogen bond, or covalent bond (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder reaction, etc.) with a target material.

[0030] Complex: A carrier, such as a solid support, and a sequenceable nucleic acid fragment attached to the carrier. In some of the examples disclosed herein, the carrier also includes one member of a biotin-streptavidin binding pair, the other member of which is part of the capture site.

[0031] Deposition: Any suitable application technique, which may be manual or automated, and in some cases results in modification of surface properties. Generally, deposition can be performed 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.

[0032] 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.

[0033] Desthiobiotin: A sulfur-free biotin analog that binds less tightly to avidin and streptavidin than biotin. The term may also include dual desthiobiotin and triple desthiobiotin.

[0034] Each: When used in reference to a collection of items, each identifies an individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where explicit disclosure or context clearly dictates otherwise.

[0035] Flow cell: A container having a chamber (e.g., which may include a flow channel) in which a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some examples, the chamber allows for detection of a 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.

[0036] 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.

[0037] Fragment: A portion or piece of genetic material (e.g., DNA, RNA, etc.). Contiguous library fragments are smaller pieces of a longer nucleic acid sample that have been fragmented, with the contiguous information of the longer nucleic acid sample preserved in the fragment.

[0038] 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.

[0039] A "template" nucleic acid molecule (or strand) can refer to the sequence that is to be analyzed.

[0040] 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).

[0041] Primer: A nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a library fragment. As an example, an amplification primer can serve as a starting point for template amplification and cluster generation. As another example, a synthesized nucleic acid (template primer) 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 primer can be any number of bases long 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.

[0042] 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 solid support index, and the other end (containing the P7 or P7' sequence) can contain a sample index. Sequenable nucleic acid fragments can be attached via transposon insertion, where the inserted DNA molecule is immobilized on the surface of a 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.

[0043] Sequencing surface: Comprises a polymeric hydrogel having one or more types of amplification primers grafted thereto. The sequencing surface may also include chemical capture agents for immobilizing complexes at or near the amplification primers.

[0044] 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 recognized particle shape, whether having regular or irregular dimensions. The solid support can have a sequencing library attached to it.

[0045] Target material: Any substance that contains a biotin-streptavidin bond or a desthiobiotin-streptavidin bond.

[0046] Transferred and non-transferred strands: The term "transferred strand" refers to a sequence comprising the transferred portion of two hybridized transposon ends. Similarly, the term "non-transferred strand" refers to a sequence comprising the non-transcribed portion of two hybridized transposon ends. The 3' end of the transferred strand is ligated or transferred into a double-stranded fragment in an in vitro transposition reaction. The non-transferred strand, which exhibits a transposon end sequence complementary to the transferred transposon end sequence, is not ligated or transferred into a double-stranded fragment in an in vitro transposition reaction.

[0047] Transposase: An enzyme that forms a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and can catalyze the insertion or transposition of the transposon end-containing composition into a double-stranded DNA sample in which it is incubated, for example, in an in vitro transposition reaction. Transposases provided herein can also include integrases from retrotransposons and retroviruses. While many examples described herein refer to Tn5 transposase and / or hyperactive Tn5 transposase, it is understood that any transposition system capable of inserting transposon ends with sufficient efficiency to 5' tag and fragment a DNA sample for the intended purpose can be used. In certain examples, the transposition system can insert transposon ends in a random or near-random manner and 5' tag and fragment a DNA sample.

[0048] Transposome complex: A complex formed between an integration enzyme (e.g., integrase or transposase) and a nucleic acid containing an integration recognition site (e.g., a transposase recognition site). For example, a transposome complex can be a transposase enzyme preincubated with double-stranded transposon DNA under conditions that support non-covalent complex formation. The double-stranded transposon DNA can include, for example, Tn5 DNA, a portion of Tn5 DNA, a transposon end composition, a mixture of transposon end compositions, or other double-stranded DNA that can interact with a transposase, such as a hyperactive Tn5 transposase.

[0049] Transposon end: A double-stranded nucleic acid DNA that exhibits only the nucleotide sequence ("transposon end sequence") necessary to form a complex with a transposase or integrase enzyme that functions in an in vitro transposition reaction. In some examples, the transposon end can form a functional complex with a transposase in a transposition reaction. For example, the transposon end can include a 19 base pair (bp) outer end ("OE") transposon end, an inner end ("IE") transposon end, or a "mosaic end" ("ME") transposon end recognized by wild-type or mutant Tn5 transposase. The transposon end can include any nucleic acid or nucleic acid analog suitable for forming a functional complex with a transposase or integrase enzyme in an in vitro transposition reaction. For example, the transposon end can include DNA, RNA, modified bases, unnatural bases, modified backbones, and can include nicks in one or both strands. Although the term "DNA" may be used in this disclosure in reference to the composition of the transposon ends, it should be understood that any suitable nucleic acid or nucleic acid analog may be utilized in the transposon ends.

[0050] cutting composition

[0051] A first example of a cleavage composition disclosed herein is suitable for disrupting biotin-streptavidin interactions at temperatures below 70°C.

[0052] A first example of a cleavage composition includes about 10% to about 50% by volume of formamide reagent, with the remainder being a salt buffer.

[0053] The formamide reagent is formamide: [ka] , which is also known as methanamide. In some examples, the formamide reagent may comprise formamide alone (without any other components). Thus, in some examples, the formamide reagent is 100% formamide. In other examples, the formamide reagent may comprise formamide and an optional buffer.

[0054] The remainder of the first example cleavage composition is a salt buffer. Any suitable salt buffer may be used. Illustratively, the salt buffer is an aqueous solution containing sodium chloride, sodium citrate, or a combination thereof. In one specific example, the salt buffer contains about 0.75 M (750 mM) sodium chloride and about 75 mM sodium citrate in water. In some examples, the salt buffer contains water having about 0.5 M sodium chloride to about 3 M sodium chloride, and / or about 50 mM sodium citrate to about 300 mM sodium citrate.

[0055] The salt buffer may also include a biocompatible surfactant. Examples of suitable biocompatible surfactants are polyethylene glycol sorbitan monolaurate or polysorbate 20 (commercially available as TWEEN™ 20 by Sigma-Aldrich). In some examples, the salt buffer further includes about 0.25% to about 1.5% by weight (based on the total weight of the salt buffer) of a biocompatible surfactant.

[0056] A first example cleavage composition comprises about 10% to about 50% by volume of formamide reagent, with the remainder being a salt buffer. Thus, the amount of salt buffer (and its individual components) in the first example cleavage composition depends on the amount of formamide reagent present. For example, the first example cleavage composition may comprise about 10% by volume of formamide reagent and about 90% by volume of salt buffer; or about 20% by volume of formamide reagent and about 80% by volume of salt buffer; or about 30% by volume of formamide reagent and about 70% by volume of salt buffer; or about 40% by volume of formamide reagent and about 60% by volume of salt buffer; or about 50% by volume of formamide reagent and about 50% by volume of salt buffer. If a larger amount (greater than 50%) of formamide reagent is included, the released library fragments hybridized to the amplification primers may denature at 25°C, which is undesirable.

[0057] In some examples, the first example of the cleavage composition comprises about 10% to about 50% by volume of a formamide reagent comprising formamide and an optional buffer, with the remainder comprising sodium chloride, sodium citrate, and a salt buffer comprising a biocompatible surfactant. In this example, the first example of the cleavage composition does not include any other components.

[0058] A second example of a cleavage composition disclosed herein is suitable for disrupting desthiobiotin-streptavidin interactions at temperatures ranging from about 60°C to about 70°C.

[0059] A second example of a cleavage composition includes free biotin and a salt buffer.

[0060] The amount of free biotin in the second example cleavage composition depends, in part, on the amount of desthiobiotin-streptavidin interactions to be disrupted by the cleavage composition. In one example, free biotin may be present in an amount ranging from about 10 molar to about 100 molar greater than the amount of desthiobiotin-streptavidin interactions to be disrupted. In one specific example, free biotin is present in the second example cleavage composition at a concentration ranging from about 2.5 μM to about 10 mM. In another example, free biotin is present in the second example cleavage composition at a concentration ranging from about 4 μM to about 8 mM.

[0061] The remainder of the second example cleavage composition is a salt buffer. Any suitable aqueous solution containing a salt may be used. In some examples, the salt is sodium chloride, sodium citrate, or a combination thereof. The salt buffer has a relatively high salt concentration. For example, the salt buffer contains about 0.75 M salt to about 0.85 M salt in water. In one specific example, the salt buffer contains about 0.75 M (750 mM) sodium chloride and about 75 mM sodium citrate in water.

[0062] The salt buffer may also include a biocompatible surfactant. Examples of suitable biocompatible surfactants include polyethylene glycol sorbitan monolaurate or polysorbate 20 (commercially available as TWEEN™ 20 by Sigma-Aldrich). In some examples, the salt buffer further includes about 0.1% to about 1% by weight of a biocompatible surfactant.

[0063] In some instances, the second example cleavage composition comprises free biotin and a balance salt buffer comprising sodium chloride, sodium citrate, and a biocompatible surfactant, in this instance, the second example cleavage composition does not include any other components.

[0064] Target materials containing biotin-streptavidin or desthiobiotin-streptavidin bonds

[0065] In some examples disclosed herein, the first cleavage composition may be used with any target material that contains a biotin-streptavidin bond to be disrupted. In other examples disclosed herein, the second cleavage composition may be used with any target material that contains a desthiobiotin-streptavidin bond to be disrupted. In any of these examples, the target material may be a complex.

[0066] Some examples of complexes 10A and 10B are shown in Figures 1A and 1B, respectively. In some examples of the methods disclosed herein, complexes 10A, 10B include solid support 12 and DNA library fragments 14, 14', 14" attached to solid support 12 via biotin-streptavidin bonds. In other examples of the methods disclosed herein, complexes 10A, 10B include solid support 12 and DNA library fragments 14, 14', 14" attached to solid support 12 via desthiobiotin-streptavidin bonds.

[0067] The solid support 12 can be, but is not limited to, glass (e.g., controlled pore glass beads), a magnetically responsive material, a plastic, such as acrylic, polystyrene, or a copolymer of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (TEFLON® from The Chemours Co); a polysaccharide or cross-linked polysaccharide, 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.

[0068] A "magnetically responsive" material responds to a magnetic field. Examples of magnetically responsive solid supports include or are composed of magnetically responsive materials. 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 Examples of magnetically responsive materials include metal oxides such as CoO, NiO, MnO, CrO, and CoMnP. One commercially available example is ThermoFisher Scientific's DYNABEADS™ M-280 Streptavidin (streptavidin-coated superparamagnetic beads). 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 the form of a bead and coated with a passivating material such as silicon oxide or silicon nitride.

[0069] Any example of solid support 12 may have the form of a solid bead, a porous bead, or a hollow bead.

[0070] Although not shown in FIG. 1A or 1B, solid support 12 is functionalized with one member of a biotin-streptavidin binding pair or a desthiobiotin-streptavidin binding pair. A "binding pair" generally refers to two agents (e.g., materials, molecules, moieties) that can be attached to one another. In some examples disclosed herein, the binding pair includes streptavidin and biotin. In other examples disclosed herein, the binding pair includes streptavidin and desthiobiotin. The streptavidin of the binding pair may be disposed on the surface of solid support 12, and the biotin or desthiobiotin of the binding pair (each of which is represented by reference numeral 20) may be attached to a DNA library fragment 14, 14', 14".

[0071] In some examples, the streptavidin on the solid support 12 may be multifunctional, in that it can bind (i) to biotin or desthiobiotin 20 attached to the DNA library fragments 14, 14', 14" and (ii) to biotin or desthiobiotin capture sites on the sequencing surface of a flow cell. In other examples, the solid support 12 may be functionalized with two different binding pair members, for example, (i) streptavidin (capable of binding to biotin or desthiobiotin 20 attached to the DNA library fragments 14, 14', 14") and (ii) another member (capable of binding to capture sites on the sequencing surface of a flow cell).

[0072] Functionalization of solid support 12 may include coating solid support 12 with streptavidin, either alone or in combination with another binding pair member.

[0073] DNA library fragments 14, 14', 14" are attached to solid support 12. Each DNA library fragment 14, 14', 14" comprises a long portion of genetic material (e.g., fragment 16, 16', 16") with adapters (e.g., 18, 18', 18", 22, 22', 22") at the 3' and 5' ends. DNA library fragments 14, 14', 14" may be prepared using any library preparation technique that fragments longer genetic material and incorporates desired adapters at the ends of the fragments. Some suitable library preparation techniques are described with reference to Figures 1A and 1B. However, it should be understood that other library preparation techniques may also be used.

[0074] 1A shows an example of a complex 10A containing DNA library fragments 14, 14'. These DNA library fragments 14, 14' are sequenceable because they contain fragments 16, 16' (from a larger nucleic acid sample) and adaptors 18, 22 or 18', 22' at opposite ends of fragments 16, 16'. The contiguity of fragments 16, 16' is preserved on solid support 12.

[0075] Although exemplary methods for producing complex 10A are described herein, it should be understood that other methods may be used, provided that the sequenceable nucleic acid fragments 14, 14' are attached to the solid support 12 via a biotin-streptavidin binding pair or a desthiobiotin-streptavidin binding pair.

[0076] In one example of a method for forming the complex 10A shown in FIG. 1A, the adapter sequence 18, 18' is conjugated to biotin or desthiobiotin 20. In one example, the adapter sequence 18, 18' may include a first sequence (P5') 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. 3A, 3B, and 3C). The adapter sequence 18, 18' may also include an index sequence or barcode sequence. The adapter sequence 18, 18' is conjugated to biotin or desthiobiotin 20, which can then be bound to the surface of a solid support 12 containing streptavidin from a biotin-streptavidin or desthiobiotin-streptavidin binding pair.

[0077] In this example, transposome complexes (not shown) may also be bound to the solid support 12 at the start of the library preparation method. Before loading the transposome complexes onto the solid support 12, partial Y adapters may be mixed with a transposase enzyme (e.g., two Tn5 molecules) to form transposome complexes. The partial Y adapters may contain two mosaic end sequences (or other transposon end sequences) that hybridize to each other. One of the mosaic end sequences is called a free mosaic end sequence because it has two free ends. For example, one end can be attached to adapters 18, 18', and the other end can be attached to fragmented DNA strands 16, 16' during tagging. This mosaic end sequence is part of the transferred strand. The other 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 sequence 22, 22' is not attached to the fragmented DNA strand 16, 16' during tagging and is therefore part of the non-transferred strand.

[0078] Loading the transposome complexes onto the solid support 12 may include mixing the transposome complexes with the solid support 12 and exposing the mixture to suitable conditions for ligating the free ends of the free mosaic ends to the 3' ends of the adapter sequences 18, 18'. Individual transposome complexes may be attached to each of the adapter sequences 18, 18' on the solid support 12.

[0079] Next, in this example 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 support 12 with adapter sequences 18, 18' and transposome complexes bound thereto. When the sample comes into contact with 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 ligating the other free end of a free mosaic (or other transposon) end sequence). Sequential tagging of the longer nucleic acid sample results in multiple bridge molecules between transposome complexes. The bridge molecules wrap around solid support 12. The transposome complexes maintain the proximity of the longer nucleic acid sample as bridge molecules.

[0080] The transposase enzyme can then be removed by sodium dodecyl sulfate (SDS) treatment or by heat or proteinase K digestion. Removal of the transposase enzyme leaves fragments 16, 16' attached to the solid support 12.

[0081] To complete the sequenceable DNA library fragments 14, 14', further extension and ligation is performed to ensure that the sample fragments 16, 16' are attached to the adapter sequences 22 and 22'. The resulting complex 10A is shown in Figure 1A.

[0082] Each sequenceable DNA library fragment 14, 14' comprises a contiguous library fragment 16, 16' with a respective adapter sequence 18 and 22 or 18' and 22' attached to either end. The adapter sequence 18, 18' is initially attached to the solid support 12 and comprises a first sequence complementary to a first sequencing primer sequence and one of the flow cell primers. The adapter sequence 18, 18' is attached to the biotin or desthiobiotin of a biotin-streptavidin or desthiobiotin-streptavidin 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 DNA library fragment 14, 14' contains an adapter suitable for amplification (e.g., bridge amplification) and sequencing, PCR amplification is not performed. Thus, these fragments 14, 14' are 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 and the library fragments 14, 14' may be suitable for linked long read applications.

[0083] FIG. 1B shows another complex 10B including a solid support 12 and sequenceable DNA library fragments 14" attached to the solid support 12 using a biotin-streptavidin binding pair or a desthiobiotin-streptavidin binding pair. In one example, a PCR-free nucleotide library is created in a tube, and the library is then hybridized to the solid support 12 in the tube. In the example shown in FIG. 1B, adaptors 18", 22" are added to the library fragments 16" in the tube, a primer having biotin or desthiobiotin 20 attached thereto is hybridized to the adaptor 18" in the tube, and the sequenceable nucleic acid fragments 14" are then bound to the solid support 12 via the biotin-streptavidin binding pair or the desthiobiotin-streptavidin binding pair. In another example, the solid support 12 may have primers attached to the support 12 via a biotin-streptavidin binding pair (e.g., streptavidin on the support 12 and biotin or desthiobiotin 20 attached to the primers). These primers hybridize to adapters 18" attached to the library fragments 16, 16' (thus the primers and biotin or desthiobiotin 20 are at one end of the fragments 16, 16' 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 adapters, as shown in Figure 1B).

[0084] As previously mentioned, other library preparation techniques may be used, so long as the DNA library fragments 14, 14', 14'' are attached to the solid support 12 via a biotin-streptavidin or desthiobiotin-streptavidin binding pair.

[0085] Library preparation beads with dual release mechanisms

[0086] In some examples disclosed herein, the DNA library fragments 16, 16', 16" are attached to the solid support 12 and are releasable from the solid support 12 with a dual release mechanism. The dual release mechanism includes a cleavage site and a splint, each of which is described in more detail with reference to Figures 2A-2C.

[0087] An example of a library preparation bead 11 before tagging is shown in Figure 2A. The library preparation bead 11 includes a solid support 12 and a transposome complex 52 attached to the solid support 12, the transposome complex 52 including (i) a transposase enzyme 54, (ii) a double-stranded molecule 56 bound to the transposase enzyme 54, the double-stranded molecule 56 including: (iia) a transferred strand 58 comprising a 3' transposon end sequence 62A, an adapter sequence 18, a cleavage site 64, and a 5' ligated end sequence 66, where the adapter sequence 18 and the 5' ligated end sequence 66 are adjacent to the cleavage site 64; and (iib) a non-transferred strand 60 comprising a 3' transposon end sequence 62B; and (iii) a splint sequence 68 hybridized to at least a portion of the adapter sequence 18 and at least a portion of the 5' ligated end sequence 66 so as to splint the cleavage site 64.

[0088] The solid support 12 may be any of the examples described herein.

[0089] As described above, the transposome complex 52 includes a transposase enzyme 54. The transposase enzyme 54 may be any of the examples described herein. In the example shown in FIG. 2A, the transposome 54 includes a dimer (e.g., Tn5) having respective monomers A and B bound to a double-stranded molecule 56. Thus, this example of the transposome complex 52 includes the transposase enzyme 54 and two double-stranded molecules 56 bound to the monomers A and B of the transposase enzyme 54, respectively. Each double-stranded molecule 56 includes hybridized 3' transposon end sequences 62A and 62B. The 3' transposon end sequences 62A and 62B are complementary to each other and contain only the nucleotide sequences necessary to form a complex with the transposase enzyme 54. Thus, the monomers A and B bind to the double-stranded molecule 56 via their hybridized 3' transposon end sequences 62A and 62B, respectively.

[0090] The 3' transposon end sequence 62A of the double-stranded molecule 56 constitutes part of the transferred strand 58. In addition to the 3' transposon end sequence 62A, the transferred strand 58 also includes an adapter sequence 18 linked to the 3' transposon end sequence 62A, a cleavage site 64 linked to the adapter sequence 18, and a 5' ligated end sequence 66 linked to the cleavage site 64. Thus, the adapter sequence 18 and the 5' ligated end sequence 66 are adjacent to the cleavage site 64.

[0091] The adapter sequence 18 may be any of the embodiments disclosed herein. If amplification is performed on the surface of a flow cell, the adapter sequence 18 may include a first sequencing primer sequence (e.g., read 1 sequencing primer sequence), a first sequence (e.g., P5') complementary to at least a portion of one of the amplification primers (e.g., P5) on the flow cell, and / or an index or barcode sequence.

[0092] The cleavage site 64 is selected from the group consisting of a chemically cleavable cleavage site, an enzymatically cleavable cleavage site, and a photocleavable cleavage site.

[0093] In some examples, the cleavage site 64 is selected from the group consisting of chemically cleavable cleavage sites and enzymatically cleavable cleavage sites. The chemically cleavable cleavage site may comprise a chemically cleavable nucleobase, a chemically cleavable modified nucleobase, or a chemically cleavable linker (e.g., between nucleobases). Examples of chemically cleavable nucleobases, modified nucleobases, or linkers include vicinal diols (e.g., 1,2-diols cleavable by periodate), disulfides, silanes, azobenzenes, photocleavable groups, allyl T (thymine nucleotide analogs with allyl functionality), allyl ethers, or azide-functional ethers. The enzymatically cleavable cleavage site may be an enzymatically cleavable nucleobase. The enzymatically cleavable nucleobase may be susceptible to cleavage by reaction with glycosylases and endonucleases or by reaction with exonucleases. One specific example of an enzymatically cleavable nucleobase is deoxyuracil (dU), which can be targeted by the USER enzyme (a mixture of DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII). Other abasic sites may also be used. Another specific example of an enzymatically cleavable nucleobase is RNA, which can be targeted by RNase.

[0094] In another example, the cleavage site 64 may be a photocleavable site. A photocleavable site may be susceptible to cleavage upon exposure to light of a particular wavelength. An example of a photocleavage site is a nitrobenzyl linker having the following structure: [ka] It is cleaved when irradiated with 365 nm light.

[0095] 5'-linked terminal sequence 66 may be a nucleotide sequence or a binding pair member that can be attached to the surface of solid support 12. In some examples, solid support 12 may have a reactive group on its surface for covalent attachment to 5'-linked terminal sequence 66. Examples of such reactive groups include carboxylic acids, primary aliphatic amines, aromatic amines, aromatic chloromethyls (e.g., vinylbenzyl chloride), amides, hydrazides, aldehydes, hydroxyls, thiols, and epoxies. These reactive groups may be inherently present on the surface of solid support 12 or may be incorporated into the surface of solid support 12 through any suitable functionalization technique (e.g., chemical reaction, coating solid support 12 with a reactive group-containing polymer, etc.). In other examples, solid support 12 may be coated with one member of a binding pair, and 5'-linked terminal sequence 66 may be attached to the other member of the binding pair.

[0096] Splint sequence 68 is a nucleotide sequence having a portion complementary to at least a portion of adapter sequence 18 and a portion complementary to at least a portion of 5' linkage end sequence 66. Each portion of splint sequence 68 hybridizes to a respective portion of adapter sequence 18 and 5' linkage end sequence 66 such that splint sequence 68 bridges cleavage site 64. The portion of splint sequence 68 that splints or bridges cleavage site 64 is not attached to cleavage site 64. Although not shown in Figures 2A-2C, some examples of splint 68 may include a portion complementary to the entire 5' linkage end sequence 66. In this example, the 3' end of splint 68 may be attached to solid support 12 via a bond pair or a covalent bond.

[0097] The 3′ transposon end sequence 62 B of the double-stranded molecule 56 constitutes the entire non-transferred strand 60 .

[0098] FIG. 2B shows a schematic diagram of the tagging process, including library preparation beads 11. A DNA sample 70 may be mixed with the library preparation beads 11. The DNA sample 70 may be DNA or complementary DNA (cDNA) derived from an RNA sample. Conversion of the RNA sample to a cDNA sample may be performed using reverse transcription utilizing a reverse transcriptase. In some examples, a kit for reverse transcription and second strand synthesis is used. In these examples, a high-capacity cDNA reverse transcription kit from ThermoFisher Scientific may 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 may be used.

[0099] The DNA sample 70 may be incorporated into a carrier liquid (e.g., tagging buffer) to generate a library preparation fluid. This library preparation fluid may be mixed with library preparation beads 11. Transposome complexes 52 bind to the sample DNA 70 and generate two nicks in the DNA backbone, indicated by lightning bolts in FIG. 2B. This creates fragments 16 and 16'. In the example shown in FIG. 2B, the nicks are 9 bases apart on either strand. It should be understood that transposome complexes 52 can generate gaps of 7, 8, 9, 10, 11, or 12 bp between the nicks. One of the two strands of each transposome complex 52 (e.g., transferred strand 58) is ligated to the 5' end of each fragment 16 and 16' at each nick location. The attachment of transferred strand 58 to each fragment 16 and 16' is shown in FIG. 2C.

[0100] The transposase enzyme 54 (both monomers A and B) may then be removed by sodium dodecyl sulfate (SDS) treatment or heat or proteinase K digestion. Removal of the transposase enzyme 54 leaves fragments 16, 16' attached to the solid support 12 via the transferred strand 58.

[0101] To complete the formation of sequenceable fragments 14, 14' (including adapters 18, 22 at both ends similar to the example shown in FIG. 1A), further extension and ligation is performed to ensure that sample fragments 16, 16' are attached to non-transferred strand 60 and / or additional adapter sequence 22. In some cases, non-transferred strand 60 is removed (e.g., via heat or enzymatic digestion), and then adapter sequence 22 is added to the 3' end of fragments 16, 16' (e.g., via extension ligation). In other cases, non-transferred strand 60 is ligated to the 3' end of fragments 16, 16', and then adapter 22 is added to the now-ligated non-transferred strand 60.

[0102] If it is desired to release the sequenceable fragments 14, 14' from the solid support 12, a dual release mechanism may be used as further described herein with reference to FIG.

[0103] Flow cell

[0104] In some examples, complexes 10A, 10B, or prepared library preparation beads 11' (shown in FIG. 4) are introduced into a flow cell for amplification and sequencing. A top view of an example flow cell 24 is shown in FIG. 3A. As discussed with reference to FIGS. 3B and 3C, some examples of flow cell 24 include two opposing sequencing surfaces. An example of an unpatterned sequencing surface 30, 30' is shown in FIG. 3B, and an example of a patterned sequencing surface 32, 32' is shown in FIG. 3C. Each sequencing surface 30, 30' or 32, 32' is supported by a substrate (shown generally as 26 in FIG. 3A), and a flow channel (shown generally as 28 in FIG. 3A) is defined between sequencing surfaces 30, 30' or 32, 32'. In other examples, flow cell 24 includes one sequencing surface supported by a substrate and a lid attached to the substrate. In these examples, a flow channel 28 is defined between the sequencing surface 30 or 32 and the lid.

[0105] In any of these examples, the substrate 26 may be a single layer / material. Examples of single layer substrates are shown in FIG. 3B as reference numerals 26A and 26A'. Examples of suitable single layer substrates 26A, 26A' include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefin / cycloolefin polymer (COP) (such as ZEONOR® from Zeon), polyamide, 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.

[0106] The substrate 26 may also be a multilayer structure. Examples of multilayer substrates are shown in FIG. 3C with 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 FIG. 3C, 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.

[0107] In one example, 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 (about 3 meters). In one example, substrate 26 is a wafer having a diameter ranging from about 200 mm to about 300 mm. In another example, substrate 24 is a die having a width ranging from about 0.1 mm to about 10 mm. While exemplary dimensions are provided, it should be understood that substrate 26 having any suitable dimensions can be used. As another example, a panel may be used that is a rectangular support having a surface area greater than a 300 mm round wafer.

[0108] In the example shown in FIG. 3A , 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 some of the embodiments disclosed herein, each flow channel 28 is a region defined between two sequencing surfaces (e.g., 30 and 30′ or 32 and 32′) and two attached substrates (e.g., 26A and 26A′ or 26B and 26B′). In other examples of the embodiments disclosed herein, each flow channel 28 is a region defined between one sequencing surface (e.g., 30 or 32) and a lid. Fluids described herein can be introduced into and removed from the flow channels 28. 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.

[0109] The 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, the portion of the flow channel 28 is etched into the glass substrate 26. In another example, the portion of the flow channel 28 may be patterned into the resin 36, 36' of the multilayer substrate 28B, 28B' using photolithography, nanoimprint lithography, or the like. In yet another example, a separate material (e.g., material 50 in FIGS. 3B and 3C) may be applied to the substrate 26, the separate material defining at least a portion of the wall of the flow channel 28.

[0110] In one example, the flow channels 28 have a rectilinear configuration. The length and width of the flow channels 28 may be less 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).

[0111] The depth of each flow channel 28 can be as small as several monolayers, for example, when using microcontact, aerosol, or inkjet printing to deposit separate materials 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 greater. 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 a patterned sequencing surface 32, 32′ is used.

[0112] 3B 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.

[0113] 3B, a portion of the flow channel 28 is defined in each of the single-layer substrates 26A, 26A'. For example, each substrate 26A, 26A' may have a recessed region 38, 38' defined therein into which components of the sequencing surface 30, 30' may be introduced. It should be understood that any space within the recessed region 38, 38' that is not occupied by components of the sequencing surface 30, 30' may be considered to be part of the flow channel 28.

[0114] 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'.

[0115] 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 each p is an integer ranging from 1 to 50; n is an integer ranging from 1 to 50,000, and and m is an integer ranging from 1 to 100,000.

[0116] 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).

[0117] 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 specific example, can be about 312 kDa.

[0118] 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.

[0119] 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] can be replaced by R D , R E , and R F are each H or C1-C6 alkyl, and R G and R H is each a C1-C6 alkyl (rather than H as in acrylamide). In this example, q may be an integer ranging from 1 to 100,000. In another example, in addition to the acrylamide units, N,N-dimethylacrylamide may be used. In this example, structure (I) contains, in addition to the repeating features "n" and "m", [ka] and R D , R E , and R F are each H or C1-C6 alkyl, and R G and R H is each C1-C6 alkyl. In this example, q can be an integer ranging from 1 to 100,000.

[0120] 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 having 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 comprising 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- to 10-membered rings that exist as single ring structures or fused structures. Some specific examples of Z include pyrrolidinyl, pyridinyl, or pyrimidinyl.

[0121] As yet another example, the polymer hydrogel 40, 40′ may include repeat units of each of structures (III) and (IV): [ka] 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 each independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0122] It should be understood that other molecules can 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-shaped or star-block polymers, branched polymers including dendrimers, etc. For example, monomers (such as acrylamide) can be incorporated into the branches (arms) of a star polymer, either randomly or in blocks.

[0123] 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'.

[0124] 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 to generate surface-activating agents (e.g., -OH groups) that can attach to the polymer hydrogel 40, 40'.

[0125] 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 18°C ​​to about 25°C) to about 95°C for a time period ranging from about 1 millisecond to about several days.

[0126] Polishing may then 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'.

[0127] The sequencing surface 30, 30' also includes amplification primers 42, 42' attached to a polymeric hydrogel 40, 40'.

[0128] 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 attachment at or near the 5′ end of the primer 42, 42′. This attachment (i) frees the adapter-specific portion of the primer 42, 42′ to anneal to its cognate sequenceable nucleic acid fragment and (ii) frees the 3′ hydroxyl group for primer extension. Any suitable covalent bond can be used for this purpose. Examples of terminal primers that can be used include alkyne-terminated primers (e.g., which can be attached to an azide surface moiety of the polymer hydrogel 40, 40′) or azide-terminated primers (e.g., which can be attached to an alkyne surface moiety of the polymer hydrogel 40, 40′).

[0129] 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 the P5 or P7 primer can be grafted onto each of the polymer hydrogels 40, 40'.

[0130] 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′.

[0131] In the example shown in FIG. 3B, the chemical capture sites 44, 44′ include 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 may be used. In some examples, the chemical capture agent may be biotin, which may serve to attach the streptavidin-coated complex 10A, 10B to the flow cell sequencing surface 30, 30′ or 32, 32′. In other examples, the chemical capture agent may be another member of a binding pair other than the biotin-streptavidin binding pair. In these other examples, the complex 10A, 10B includes two different binding pair members, for example, (i) streptavidin (capable of binding to the biotin 20 attached to the DNA library fragments 14, 14′, 14″) and (ii) another member (capable of binding to the chemical capture agent of the capture site 44, 44′ on the sequencing surface of the flow cell). In these other examples, the chemical capture agent may be a non-biotin member of a binding pair, the other member of which is attached to solid support 12 in addition to streptavidin.

[0132] 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 (e.g., biotin or another member of a binding pair) attached to them. These complementary primers can be hybridized to several amplification primers 42, 42′ to form chemical capture sites 44, 44′.

[0133] In another example, the chemical scavenger may be deposited at the desired location 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 scavenger will be deposited, thus forming the chemical capture sites 44, 44'. The chemical scavenger may then be deposited (e.g., via lift-off, dissolution, or another suitable technique), and the mask removed. In this example, the chemical capture sites 44, 44' may comprise a monolayer or thin layer of the chemical scavenger.

[0134] 3C 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 substrates 26B, 26B′, which can then be attached to one another (e.g., via material 50) to form the example flow cell 24.

[0135] 3C, 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'.

[0136] 3C, 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'.

[0137] 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.

[0138] 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-based resins, non-polyhedral oligomeric silsesquioxane 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.

[0139] As used herein, the term "polyhedral oligomeric silsesquioxane" (commercially available from Hybrid Plastics under the trademark POSS®) refers to a hybrid intermediate between silica (SiO2) and silicone (R2SiO) (e.g., RSiO 1.5 ) refers to a chemical composition having the formula [RSiO 3 / 2 ] n wherein the R groups can 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 can contain one or more different cage or core structures as monomer units. The average cage content can be adjusted during synthesis and / or controlled by purification methods, and a distribution of cage sizes of the monomer units can be used in the embodiments disclosed herein.

[0140] As shown in FIG. 3C , 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 stripes, swirls, lines, triangles, rectangles, circles, arcs, ticks, checkerboards, diagonals, arrows, squares, and / or crosshatches.

[0141] 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 may be present at a density of about 2 million per mm. 2 Approximately 100, 1mm per 2 Approximately 1,000 per 1mm 2 Approximately 100,000 per 1mm 2 Approximately 1 million per 1mm 2 Approximately 2 million per 1mm 2 Approximately 5 million per 1mm 2 Approximately 10 million per mm 2The density of the depressions 48, 48' may be adjusted to different densities, including densities of about 50 million per depression. It should further be understood that the density of the depressions 48, 48' in the patterned material 36, 36' may be between one of the low and high values ​​selected from the ranges above. By way of example, a high-density array may be characterized as having depressions 48, 48' separated by less than about 100 nm, a medium-density array may be characterized as having depressions 48, 48' separated by about 400 nm to about 1 μm, and a low-density array may 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' may 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.

[0142] The layout or pattern of the depressions 48, 48′ may also or alternatively be characterized in terms of average pitch, or the spacing from the center of a depression 48, 48′ to the center of an adjacent depression 48, 48′ (center-to-center spacing) or the spacing from the edge of one depression 48, 48′ to the edge of an adjacent depression 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 depressions 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.

[0143] The size of each recess 48, 48' may be characterized by its volume, open area, depth, and / or diameter.

[0144] Each well 48, 48' may have any volume capable of confining at least some of the fluid introduced into flow cell 24. The minimum or maximum volume may be selected to correspond, for example, to the expected throughput (e.g., multiplexing), resolution, nucleotide, or analyte reactivity for downstream use of flow cell 24. For example, the volume may be at least about 1 x 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.

[0145] The area occupied by each recess opening can be selected based on the same criteria as 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 -2μm 2 The area occupied by the opening of each recess may be greater than, less than, or in between the above values.

[0146] The depth of each depression 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 may be about 100 μm, up to about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each depression 48, 48′ may be greater than, less than, or between the values ​​indicated above.

[0147] In some cases, the diameter or length and width of each depression 48, 48' may 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 may be at most about 1×10 3 The diameter or length and width of each depression 48, 48' may be greater than, less than, or between the values ​​specified above.

[0148] 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.

[0149] In the example shown in Figure 3C, 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 3B, so that the polymer hydrogel 40, 40' is present in the depressions 48, 48' and not in the surrounding interstitial regions 46, 46'.

[0150] In the example shown in Figure 3C, 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 3B and is therefore grafted onto the polymer hydrogel 40, 40' rather than the surrounding interstitial regions 46, 46'.

[0151] 3C, the chemical capture sites 44, 44' are chemical capture agents that are 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.

[0152] In other examples, the chemical capture sites 44, 44' are 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' are 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'.

[0153] Any of the examples of chemical capture agents disclosed herein may be used in the example shown in Figure 3C. The chemical capture agent may be biotin or another member of a binding pair, depending in part on how solid support 12 is functionalized.

[0154] As shown in Figures 3B and 3C, substrates 26A and 26A' or 26B and 26B' ​​are attached to one another such that sequencing surfaces 30 and 30' or 32 and 32' face one another with flow channel 28 defined therebetween.

[0155] 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).

[0156] 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 some 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.

[0157] Although not shown, it should be understood that a lid may be attached to one of substrates 26A' or 26B' ​​such that the flow cell has one sequencing surface.

[0158] Methods and kits involving target materials having biotin-streptavidin bonds

[0159] An example method utilizing the first example cleavage composition generally includes: introducing library fragments 14, 14', 14" into a flow cell 24, where the library fragments 14, 14', 14" are attached to a streptavidin-coated solid support 12; introducing a biotin-streptavidin cleavage composition into the flow cell 24, where the biotin-streptavidin cleavage composition comprises about 10% to about 50% by volume of formamide reagent, with the remainder being a salt buffer; and allowing the biotin-streptavidin cleavage composition to incubate in the flow cell at a temperature ranging from about 60°C to about 70°C, thereby releasing at least some of the library fragments 14, 14', 14" from the solid support 12 and seeding them onto the amplification primers 42, 42' on the surface of the flow cell 24.

[0160] Prior to performing the method, library fragments 14, 14', 14" may be prepared and attached to solid supports 12. In one example, complexes 10A or 10B may be prepared using a nucleic acid sample and a library preparation fluid comprising a plurality of solid supports 12.

[0161] In some examples, as described with reference to Figure 1A, each of the solid supports 12 in the library preparation fluid may have, for example, an adapter (such as adapter 18) attached thereto. Tagging and library preparation may be performed as defined in Figure 1A to form complex 10A. The nucleic acid sample 70, solid support 12, partial Y adapter, and transposase enzyme may be contained in separate fluids until it is desired to form complex 10A.

[0162] In other examples, each of the solid supports 12 in the library preparation fluid may have, for example, oligonucleotides attached to the support. In some examples, a nucleotide library preparation that does not involve PCR may be performed separately from the solid supports 12, and the prepared library fragments may then be hybridized to oligonucleotides (primers) attached via biotin 20 on the surface of the solid supports 12, an example of which is described with reference to FIG. 1B. Other examples of library preparation (e.g., including PCR) may also be used, so long as the fragments are denatured to single-stranded fragments before hybridizing to oligonucleotides bound on the solid supports 12 via biotin 20.

[0163] The library fragments 14, 14', 14'' attached to the solid support 12 (in this example, complexes 10A, 10B) may be added to a fluid. The fluid may 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 may be adjusted to allow complexes 10A, 10B to flow to the desired sequencing surface. The greater the density difference between the solid support 12 and the fluid, the faster the sedimentation time of complexes 10A, 10B. By way of example, the fluid may be Tris-HCl buffer or 0.5x saline sodium citrate (SSC) buffer.

[0164] The fluid containing complexes 10A, 10B may then be introduced into flow cell 24. Once introduced into flow cell 24, complexes 10A, 10B can attach to capture sites 44, 44' because complexes 10A, 10B and capture sites 44, 44' each contain a member of a binding pair. In some examples, the binding pair is biotin-streptavidin. Capture sites 44 and / or 44' immobilize at least some of complexes 10A, 10B.

[0165] It should be understood that some complexes 10A, 10B in the fluid may not be captured, and these complexes 10A, 10B are removed from the flow cell 24 before further processing. A predetermined time may be allowed to elapse before removing the fluid and any non-immobilized complexes 10A, 10B from the flow cell 24. In one example, the predetermined time may range from about 5 minutes to about 30 minutes to obtain the desired number of immobilized complexes 10A, 10B. Longer incubation times may also be used.

[0166] This example method then includes washing the fluid and uncaptured complexes 10A, 10B from the flow cell 24. Washing can include introducing a wash fluid into the flow cell 24. The flow may not settle, but may push any complexes 10A, 10B (or other target material) immobilized on the sequencing surface 30, 30' or 32, 32' through the outlet port of the flow cell 24. The immobilization mechanism (e.g., binding pair, hybridization, covalent bond, etc.) between the complexes 10A, 10B (or other target material) and the capture sites 44, 44' of the sequencing surface 30, 30' or 32, 32' may prevent any settled and immobilized complexes 10A, 10B (or other target material) from becoming part of the outlet flow.

[0167] The method includes introducing a first example of a cleavage composition into the flow cell 24. Any example of the first example of a cleavage composition disclosed herein may be used.

[0168] The first example of the cleavage composition is allowed to incubate in the flow cell 24 at a temperature ranging from about 60° C. to about 70° C. In one example, the temperature is about 65° C. In one example, the first example of the cleavage composition is allowed to incubate in the flow cell 24 for a time ranging from about 2 minutes to about 5 minutes. In one example, the time is about 5 minutes.

[0169] During incubation, the first example cleavage composition effectively disrupts the biotin-streptavidin bonds that hold the library fragments 14, 14', 14" to the solid support 12. Thus, the first example cleavage composition releases at least some of the library fragments 14, 14', 14" from the solid support 12. At the incubation temperature, the released library fragments 14, 14', 14" can also seed onto the amplification primers 42, 42' on the sequencing surface 30, 30' or 32, 32' of the flow cell 24. Thus, library fragment 14, 14', 14" release and seeding may be achieved with a single reagent.

[0170] The primers 42, 42' of the respective sequencing surfaces 30, 30' or 32, 32' of the flow cell 24 may be seeded with the released fragments 14, 14' or 14'. Seeding is achieved by hybridization between the first or second sequence of the adapter 18 or 22 of the fragment 14, 14' or 14'' and the complementary sequence of the primers 42, 42' of the respective sequencing surfaces 30, 30' or 32, 32'. Seeding may occur at the cleavage composition incubation temperature.

[0171] The location of seeding 14, 14', or 14" within flow cell 24 depends, in part, on how 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 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 depressions 48, 48'.

[0172] If the biotin-streptavidin binding pair is also used to capture the complexes 10A, 10B at the capture sites 44, 44', the first example of the cleavage composition can also release the solid support 12 from the capture sites 44, 44'.

[0173] Thus, in some example methods, at least some of the streptavidin-coated solid supports 12 become bound to the biotin capture sites 44, 44′ on the surface of the flow cell 24, and allowing a biotin-streptavidin cleavage composition (a first example of a cleavage composition) to incubate within the flow cell 24 similarly releases at least some of the bound streptavidin-coated solid supports 12 from the biotin capture sites 44, 44′. Removal of the solid supports 12 after seeding with fragments 14, 14′, 14″ is desirable for a clean surface for downstream clustering and sequencing.

[0174] If different binding pairs are used to capture complexes 10A, 10B at capture sites 44, 44', a separate release composition may be introduced into flow cell 24 to remove solid support 12 from capture sites 44, 44'.

[0175] This example method may then include rinsing the biotin-streptavidin cleavage composition (a first example of a cleavage composition) from the flow cell 24 after incubation, thereby removing the streptavidin-coated solid support 12 and any unseeded library fragments 14, 14', 14" from the flow cell 24. The flow may push any released solid support 12 and any unseeded library fragments 14, 14', 14" through an exit port in the flow cell 24. An immobilization mechanism (e.g., hybridization) between the fragments 14, 14', 14" on the sequencing surface 30, 30' or 32, 32' and the amplification primers 42, 42' may prevent any fragments 14, 14', 14" from becoming part of the exit flow.

[0176] A kit for carrying out this example of the method described herein may include a streptavidin-coated solid support, an adapter sequence having biotin attached to one end such that the biotin becomes attached to the streptavidin-coated solid support, a sample fluid containing a genomic sequence that has been fragmented and becomes attached to the adapter sequence, and a biotin-streptavidin cleavage composition (a first example of a cleavage composition) containing about 10% to about 50% by volume of formamide reagent and the remainder a salt buffer. The kit may also include other library preparation components, such as partial Y adapters and transposase enzymes, each of which may be contained in separate fluids until desired formation of any of the examples of complexes 10A, 10B, etc. Some examples of the kit may also include a flow cell 24.

[0177] Methods and kits involving target materials with desthiobiotinylated streptavidin binding - Patent Application 20070122999

[0178] An example method utilizing the second example cleavage composition generally includes: introducing desthiobiotinylated library fragments into a flow cell 24, where the desthiobiotinylated library fragments are attached to a streptavidin-coated solid support 12; introducing a cleavage composition into the flow cell 24, where the cleavage composition is at a temperature in the range of about 18°C ​​to about 30°C, and where the cleavage composition comprises free biotin and a salt buffer; and raising the temperature of the cleavage composition to about 60°C to about 70°C, thereby releasing at least some of the library fragments 14, 14', 14'' from the solid support 12 and seeding them onto the amplification primers 42, 42' on the surface of the flow cell 24.

[0179] Prior to carrying out this exemplary method, library fragments 14, 14', 14'' may be prepared and attached to solid support 12 as described herein with reference to Figure 1A or Figure 1B.

[0180] The library fragments 14, 14', 14" attached to the solid support 12 (i.e., complexes 10A, 10B in this example) may be added to a fluid. The fluid may be any buffered aqueous solution (e.g., a weak acid and one of its salts (conjugate base) or a weak base and one of its salts (conjugate acid).

[0181] A fluid containing complexes 10A, 10B may be introduced into flow cell 24. Once introduced into flow cell 24, complexes 10A, 10B can attach to capture sites 44, 44' because complexes 10A, 10B and capture sites 44, 44' each contain a member of a binding pair. In some examples, the binding pair is desthiobiotin-streptavidin. Capture sites 44 and / or 44' immobilize at least some of complexes 10A, 10B.

[0182] It should be understood that some complexes 10A, 10B in the fluid may not be captured, and these complexes 10A, 10B are removed from the flow cell 24 before further processing. A predetermined time may be allowed to elapse before removing the fluid and any non-immobilized complexes 10A, 10B from the flow cell 24. In one example, the predetermined time may range from about 5 minutes to about 30 minutes to obtain the desired number of immobilized complexes 10A, 10B. Longer incubation times may also be used.

[0183] This exemplary method then includes washing the fluid and uncaptured complexes 10A, 10B from the flow cell 24. Washing may include introducing a wash fluid into the flow cell 24. The flow may not settle, and any complexes 10A, 10B immobilized on the sequencing surface 30, 30' or 32, 32' may be pushed through the exit port of the flow cell 24. An immobilization mechanism between the complexes 10A, 10B (or other target material) and the capture sites 44, 44' of the sequencing surface 30, 30' or 32, 32' may prevent any settled and immobilized complexes 10A, 10B from becoming part of the exit flow.

[0184] The method includes introducing a second example of a cleavage composition into the flow cell 24. Any example of the second example of a cleavage composition disclosed herein may be used.

[0185] The second example cleavage composition is introduced at a temperature ranging from about 18° C. to about 30° C. At this temperature, and in the presence of the salt in the second example cleavage composition, desthiobiotin is stabilized and its dissociation from the solid support 12 is reduced or prevented even in the presence of free biotin. The introduced cleavage composition is allowed to settle so that fluid flow and / or mixing at least substantially ceases.

[0186] The temperature of the cleavage composition is then increased to a temperature ranging from about 60°C to about 70°C. In one example, the temperature of the cleavage composition is increased to about 65°C. At this temperature, and in the presence of free biotin, the desthiobiotin is destabilized and dissociates from the solid support 12, releasing the library fragments 14, 14', 14''. At these temperatures, the released library fragments 14, 14', 14'' can also seed onto the amplification primers 42, 42' on the sequencing surface 30, 30' or 32, 32' of the flow cell 24. Because there is no fluid flow and / or mixing when the library fragments 14, 14', 14'' are released, the released library fragments 14, 14', 14'' can diffuse and seed onto the flow cell surface at or near the solid support 12 from which they were released. Thus, both library fragment 14, 14', 14'' release and diffusion-dependent spatial seeding may be achieved with a single reagent.

[0187] If the desthiobiotin-streptavidin binding pair is also used to capture the complexes 10A, 10B at the capture sites 44, 44', the second example of the cleavage composition can also release the solid support 12 from the capture sites 44, 44'.

[0188] Thus, in some example methods, at least some of the streptavidin-coated solid supports 12 bind to biotin capture sites 44, 44' on the surface of the flow cell 24, and increasing the temperature of the previously introduced desthiobiotin-streptavidin cleavage composition (a second example of a cleavage composition) releases at least some of the bound streptavidin-coated solid supports 12 from the desthiobiotin capture sites 44, 44'. Removal of the solid supports 12 after seeding with fragments 14, 14', 14'' is desirable for a clean surface for downstream clustering and sequencing.

[0189] If different binding pairs are used to capture complexes 10A, 10B at capture sites 44, 44', a separate release composition may be introduced into flow cell 24 to remove solid support 12 from capture sites 44, 44'.

[0190] This example method may then include rinsing the desthiobiotin-streptavidin cleavage composition (a second example of a cleavage composition) from the flow cell 24 after plating, thereby removing the streptavidin-coated solid support 12 and any unseeded library fragments 14, 14', 14" from the flow cell 24. The flow may push any released solid support 12 and any unseeded library fragments 14, 14', 14" through an exit port of the flow cell 24. An immobilization mechanism (e.g., hybridization) between the fragments 14, 14', 14" on the sequencing surface 30, 30' or 32, 32' and the amplification primers 42, 42' may prevent any fragments 14, 14', 14" from becoming part of the exit flow.

[0191] A kit for carrying out this example of the method described herein may include a streptavidin-coated solid support 12, an adapter sequence having desthiobiotin attached to one end, such that the desthiobiotin becomes attached to the streptavidin-coated solid support, a sample fluid containing genomic sequences that have been fragmented and become attached to the adapter sequence, and a desthiobiotin-streptavidin cleavage composition (a second example of a cleavage composition) containing free biotin and a salt buffer. The kit may also include other library preparation components, such as partial Y adapters, a transposase enzyme, and the like, each of which may be contained in a separate fluid until it is desired to form any of the examples of complexes 10A, 10B, etc. Some examples of the kit may also include a flow cell 24.

[0192] Methods and kits involving two-stage release

[0193] As mentioned above, some examples disclosed herein utilize a dual release mechanism for releasing the sequenceable library fragments 14, 14′ from the library preparation beads 11. The dual release mechanism utilizes a cleavage site 64 and a splint 68. As described in more detail with reference to FIG. 4 , a suitable cleavage agent may be used to remove the cleavage site 64, and heat may be used to remove the splint 68. This example allows for the controlled release of the library fragments 14, 14′ because the cleavage conditions involve two orthogonal processes (one process does not initiate, affect, or otherwise interfere with the other).

[0194] The dual mechanism release may occur at the sequencing surface 30, 30' or 32, 32' of the flow cell 24, or may occur in a separate reaction vessel such as a test tube.

[0195] An example of this method is shown schematically in FIG. 4 and generally involves introducing a plurality of prepared library preparation beads 11′ into a reaction vessel (not shown), each of the prepared library preparation beads 11′ comprising a solid support 12 and a plurality of bridge molecules 72, 72′ attached to the solid support 12, each of the bridge molecules 72, 72′ comprising a double-stranded DNA fragment 16 and 16′ and a transferred strand 58, 58′ attached at its 5′ end to each strand 16, 16′ of the double-stranded DNA fragment, each transferred strand 58, 58′ comprising a 3′ transposon end sequence 62A, 62A′, a first adapter sequence 18, 18′, a cleavage site 64, 64′, and a 5′ ligation end sequence 66, 66′, wherein the first adapter sequence 18, 18′ and the 5′ ligation end sequence 66, 66′ are adjacent to the cleavage site 64, 64′. introducing second adaptors 22, 22', each attached at its 3' end to each strand 16, 16' of the double-stranded DNA fragment, and splint sequences 68, 68' hybridized to at least a portion of the first adaptor sequence 18, 18' and at least a portion of the 5' linking sequence 66, 66' so as to splint cleavage sites 64, 64'; exposing the prepared library preparation beads 11' to a cleavage agent 74 to remove the cleavage sites 64, 64', thereby leaving a plurality of bridge molecules 72, 72' attached to the solid support 12 via the splints 68, 68'; and heating the reaction vessel to a temperature at which the splints 68, 68' and the double-stranded DNA fragments 16, 16' are dissociated.

[0196] A prepared library preparation bead 11' is shown on the left side of Figure 4. Prepared library preparation bead 11' may be prepared using the methods described with reference to Figures 2A-2C. Thus, each of bridge molecules 72, 72' includes a sequenceable nucleic acid fragment 14, 14' (fragment 16, 16', a 3' transposon end sequence 62A, 62A' near the 5' end, and a respective adaptor 18, 22 or 18', 22'' at the opposite end), as well as a cleavage site 64, 64', a 5' ligation end sequence 66, 66', and a splint 68, 68'.

[0197] Although one pair of bridging molecules 72, 72' is shown on the solid support 12 in Figure 4, it should be understood that a single solid support 12 may include multiple pairs of bridging molecules 72, 72'.

[0198] To initiate the release of the sequenceable nucleic acid fragments 14, 14′ from the bridge molecules 72, 72′ and solid support 12, the method first includes exposing the prepared library preparation beads 11′ to a cleavage agent 74. The cleavage agent 74 used will depend on the cleavage site 64, 64′ of the transferred strand 58, 58′. If the cleavage site 64, 64′ is a chemical cleavage site, exposing the prepared library preparation beads 11′ to the cleavage agent 74 includes introducing the chemical cleavage agent into the reaction vessel. As an example, periodate may be used to cleave a 1,2-diol chemical cleavage site. If the cleavage site 64, 64′ is an enzymatic cleavage site, exposing the prepared library preparation beads 11′ to the cleavage agent includes introducing the enzymatic cleavage agent into the reaction vessel. As an example, a USER enzyme may be used to cleave deoxyuracil nucleotides and an RNase may be used to cleave ribonucleotides. If the cleavage site 64, 64' is a photocleavable cleavage site, exposing the prepared library preparation bead 11' to the cleavage agent 74 includes irradiating the reaction vessel with light of a wavelength that activates cleavage. As an example, a nitrobenzyl linker may be exposed to 365 nm ultraviolet light.

[0199] The incubation or light exposure of the chemical or enzymatic cleavage agent may be carried out for a time sufficient to cleave the cleavage site 64, 64'. The incubation time may depend, in part, on the reactivity of the chemical or enzymatic cleavage agent, and the light exposure time may depend on the light source (e.g., its intensity). In one example, the incubation time ranges from about 30 minutes to about 3 hours. In another example, the light exposure may range from about 30 seconds to about 2 minutes. The chemical or enzymatic cleavage agent may then be removed from the reaction vessel, or the light irradiation may be stopped. The prepared library preparation beads 11' may then be washed (e.g., with water, buffer, etc.) to remove the cleaved cleavage site 64, 64' and, in some cases, the chemical or enzymatic cleavage agent.

[0200] As shown in the center of Figure 4, after cleavage at cleavage sites 64, 64', the sequenceable fragments 14, 14' remain attached to the solid support 12. The splints 68, 68' are unaffected by the cleavage agent 74, and therefore the sequenceable fragments 14, 14' remain cross-linked and attached to the solid support 12.

[0201] To complete the release of the bridge molecules 72, 72' and the sequenceable nucleic acid fragments 14, 14' from the solid support 12, the method then includes exposing the prepared library preparation beads 11' to heat. The selected temperature is capable of dissociating the splints 68, 68' from the adaptors 18, 18' and 5' linked end sequences 66, 66', as well as dissociating any double-stranded portions of the sequenceable fragments 14, 14'. In one example, the temperature for heating ranges from about 60°C to about 70°C.

[0202] When the dual mechanism release of sequenceable library fragments 14, 14' from the prepared library preparation beads 11' occurs on the flow cell 24, at least some of the plurality of prepared library preparation beads 11' are immobilized on the surface of the flow cell 24 (e.g., via capture sites 44, 44' when introduced into the flow cell 24), and the method further includes removing the non-immobilized library preparation beads 11' from the flow cell before exposing the prepared library preparation beads 11' to the cleavage agent 74.

[0203] Additionally, spatial release of the fragments 14, 14' is achieved when dual mechanism release of the sequenceable library fragments 14, 14' from the prepared library preparation beads 11' occurs on the flow cell 24. Because the final release mechanism is thermal, there is no or minimal fluid flow and / or inertial mixing, and thus the released fragments 14, 14' can be seeded onto primers 42, 42' in proximity to the solid support 12 from which the fragments 14, 14' are released.

[0204] Sequencing

[0205] If fragment 14, 14', 14" release occurs on flow cell 24, the fragments 14, 14', 14" may be seeded onto primers 42, 42' on flow cell sequencing surface 30, 30' or 32, 32'. If fragment 14, 14', 14" release occurs in a separate reaction vessel, the released fragments 14, 14', 14" may be separated from solid support 12 (having 5' linked end sequences 66 attached thereto, as shown on the right-hand side of Figure 4), and the library fragments 14, 14', 14" may be introduced into flow cell 24 for seeding onto primers 42, 42'.

[0206] Once the fragments 14, 14', 14'' are released and seeded, the flow cell 24 is ready for downstream analysis.

[0207] The seeded library fragments 14, 14', 14'' can be amplified using cluster generation.

[0208] In one example of cluster generation, fragment 14, 14', or 14" is copied from hybridized primer 42, 42' by 3' extension using a high-fidelity DNA polymerase. The original fragment 14, 14', or 14" is denatured, and the copy remains immobilized on the sequencing surface 30, 30' or 32, 32'. Isothermal bridge amplification or some other form of amplification can be used to amplify the immobilized copy. For example, the copied template loops over and hybridizes to adjacent complementary primer 42, 42', and the polymerase copies the copied template to form a double-stranded bridge, which denatures to form two single strands. These two strands loop over and hybridize to adjacent complementary primers 42, 42' and 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 to create a dense clonal cluster. Each cluster of double-stranded bridges 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'. This example of clustering is bridge amplification, which is one example of amplification that can be performed. It should be understood that other amplification techniques, such as the Examp workflow (Illumina Inc.), can also be used.

[0209] A sequencing primer may be introduced that hybridizes to a complementary sequence on the template polynucleotide strand, making the template polynucleotide strand ready for sequencing. The 3' end of the template and any flow cell-bound primers 20 (not attached to the copy) may be blocked to prevent interference with the sequencing reaction, particularly to prevent undesired priming.

[0210] 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).

[0211] 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 complementary to the template polynucleotide strand by extending the sequencing primer. 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.

[0212] Nucleotide incorporation can be detected through an imaging event during which an illumination system (not shown) may provide excitation light to the respective sequencing surfaces 30, 30' or 32, 32'.

[0213] 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 this moiety. Thus, in instances using a reversible terminus, a deblocking reagent may be delivered to flow cell 24 after detection has occurred.

[0214] Washing 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 the detection of a sequence of length n.

[0215] In some instances, the forward strand may be sequenced and removed, and then the reverse strand may be constructed and sequenced as described herein.

[0216] 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, in which 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. In another example, the flow cell 24 disclosed herein may be used for on-cell library generation.

[0217] 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. Non-limiting examples

[0218] [Example]

[0219] Complexes similar to those shown in Figure 1A were prepared with an average diameter of 3 μm. The solid support for the complexes was ThermoFisher Scientific's DYNABEADS™ M-280 streptavidin beads. Fragments on a particular bead were derived from the same long DNA molecule (from the human genome). Library fragments were attached to the solid support via biotin oligos. Library fragments contained P5' and P7 sequences along with index sequences, and included read 1 and read 2 sequences.

[0220] The complexes were incorporated into a saline sodium citrate buffer with sodium dodecyl sulfate, and this fluid was loaded into a flow cell containing a non-patterned sequencing surface (containing P5 and P7 primers) and a lid. The non-patterned sequencing surface also contained biotin capture sites.

[0221] The flow cell was then washed with a cleaning solution.

[0222] A first example of a cleavage composition disclosed herein was prepared containing approximately 40% by volume of a formamide reagent containing formamide and trisodium citrate, and approximately 60% by volume of a salt buffer containing sodium chloride, sodium citrate, and a biocompatible surfactant. The cleavage solution was introduced into a flow cell and allowed to incubate at 80°C for approximately 20 seconds. The flow cell was then cooled to 20°C and maintained at that temperature for approximately 3 minutes.

[0223] The flow cell was then washed with a wash solution to remove the solid support and any unseeded released library fragments.

[0224] The released seeded library fragments were grown into clusters by cycle amplification. The clusters were then stained with Sytox green. The resulting images (not reproduced here) showed that the seeded library fragments were amplified in the vicinity of the complexes.

[0225] Next, sequencing was performed on the flow cell. The sequencing results showed that the long DNA molecule coverage was close to 40%. This indicated that the first embodiment of the cleavage composition disclosed herein resulted in the efficient release of library fragments from the solid support.

[0226] [Example]

[0227] Several different cleavage compositions according to the first embodiment disclosed herein were prepared containing a formamide reagent (comprising formamide and trisodium citrate) and a salt buffer (comprising sodium chloride, sodium citrate, and a biocompatible surfactant). The proportions of reagent and buffer in each cleavage composition are shown in Table 1. [Table 1]

[0228] The flow cell surface P5 and P7 primers were hybridized to TET QC primers (e.g., complementary P5 and P7 (cP5 and cP7) primers labeled with a fluorescent dye). In this example, the fluorescent label was used as a reporter of denaturation events. When the reagent caused denaturation, the fluorescently labeled primers left the surface, resulting in lower fluorescence intensity. Figure 5 shows the TET intensity (fluorescence) versus the formamide reagent and percentage used in the cleavage composition. As shown, when the formamide reagent was increased to 60% by volume, the fluorescent signal decreased, indicating that the plated library fragments were denatured. When the formamide reagent was increased to 70% by volume, the biotin-streptavidin bond could be cleaved, but the surface P5 / P7 primer hybridization was found to be unstable, even at room temperature. Therefore, the volume ratio of formamide reagent to salt buffer in the disclosed examples ranges from 1:9 to approximately 1:1 to ensure both bond cleavage and desirable seeding conditions.

[0229] [Example]

[0230] DYNABEADS™ M-280 streptavidin beads from ThermoFisher Scientific were incorporated into a saline sodium citrate buffer solution with sodium dodecyl sulfate, and this fluid was loaded into a flow cell containing a non-patterned sequencing surface (containing P5 and P7 primers) and a lid. The non-patterned sequencing surface also contained biotin capture sites. Thus, the M-280 beads were immobilized on the surface via biotin-streptavidin binding.

[0231] The unpatterned sequencing surface was imaged and the immobilized beads on the surface were counted using the microscope image.

[0232] The flow cell was then washed with a comparative cleavage agent (a formamide reagent containing formamide and trisodium citrate). The comparative cleavage agent was allowed to incubate at 65° C. for 2 minutes. The unpatterned sequencing surface was imaged, and the immobilized beads on the surface were counted using the microscope image.

[0233] The flow cell was then washed with a first example of a cleavage composition disclosed herein (referred to as an example cleavage agent), comprising approximately 50% by volume of a formamide reagent containing formamide and trisodium citrate, and approximately 50% by volume of a salt buffer containing sodium chloride, sodium citrate, and a biocompatible surfactant. The example cleavage agent was allowed to incubate at 65°C for 2 minutes. The unpatterned sequencing surface was imaged, and the immobilized beads on the surface were counted using the microscope image.

[0234] The flow cell was washed again with the example cleavage agent and imaged. The example cleavage agent was again allowed to incubate for 2 minutes at 65° C. After the second wash with the example cleavage agent, the immobilized beads on the surface were counted using microscope images.

[0235] The number of beads counted on the flow cell surface before washing, after washing with the comparative cleavage agent, after the first wash with the exemplary cleavage agent, and after the second wash with the exemplary cleavage agent is shown in Figure 6.

[0236] In the pre-wash, 100% of the beads in solution were immobilized on the flow cell surface. After washing with the comparative cleavage agent, less than 15% of the beads were removed. After the first wash with the exemplary cleavage agent, approximately 85% of the beads were removed. After the second wash with the exemplary cleavage agent, approximately 90% of the beads were removed. These results demonstrate that the exemplary cleavage mixtures disclosed herein are effective in disrupting streptavidin-biotin bonds.

[0237] [Example]

[0238] DNA fragments were attached to DYNABEADS™ M-280 streptavidin beads from ThermoFisher Scientific using desthiobiotin. Thermal release of desthiobiotinylated DNA was tested at different temperatures and under different conditions (e.g., in the presence of free biotin or free desthiobiotin).

[0239] Mixtures of free biotin and free desthiobiotin were prepared in 825 mM aqueous sodium salt solution: the first mixture contained 100 μM desthiobiotin, the second mixture 2.5 μM biotin, and the third mixture 10 μM biotin.

[0240] Control samples were exposed to either 25°C or 60°C without any mixtures. Example samples were exposed to either 25°C or 60°C in the presence of the first mixture (100 μM desthiobiotin), the second mixture (2.5 μM biotin), or the third mixture (10 μM biotin). The percentage of DNA released from the beads after each treatment was determined by quantitative PCR. The results are shown in Figure 7.

[0241] As shown in Figure 7, the majority of desthiobiotinylated DNA fragments were not released from beads in the absence of free biotin or free desthiobiotin at either 25°C or 60°C (control). Similarly, the majority of desthiobiotinylated DNA fragments were not released from beads in the presence of free desthiobiotin or free biotin (regardless of concentration) at 25°C. Beads exposed to the third mixture (10 μM free biotin) at 25°C exhibited the lowest DNA fragment release, with only approximately 1.5% of the desthiobiotinylated DNA fragments released. In contrast, approximately 90% of the desthiobiotinylated DNA fragments were released from beads in the presence of free desthiobiotin at 60°C (Mixture 1), and more than 95% of the desthiobiotinylated DNA fragments were released from beads in the presence of free biotin at 60°C (regardless of concentration; see results for Mixtures 2 and 3). Higher DNA fragment release was observed in the presence of free biotin at 60°C, at much lower concentrations than in the presence of free biotin at 25°C.

[0242] Additional Notes

[0243] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (unless such concepts are mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terms used explicitly herein, and which may also appear in any disclosures incorporated by reference, should be given the meaning most consistent with the particular concepts disclosed herein.

[0244] 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. 1. A biotin-streptavidin cleavage composition comprising: about 10% to about 50% by volume of a formamide reagent; and the balance being a salt buffer.

2. 2. The biotin-streptavidin cleavage composition of claim 1, wherein the formamide reagent is 100% formamide.

3. 3. The biotin-streptavidin cleavage composition of claim 1, wherein the salt buffer comprises about 0.75 M sodium chloride and about 75 mM sodium citrate in water.

4. 4. The biotin-streptavidin cleavage composition of claim 3, wherein the salt buffer further comprises about 0.25% to about 1.5% by weight of a biocompatible surfactant.

5. The biotin-streptavidin cleavage composition of any one of claims 1 to 4, wherein the composition comprises about 50% of the formamide reagent and about 50% of the salt buffer.

6. The biotin-streptavidin cleavage composition of any one of claims 1 to 4, wherein the composition comprises about 40% of the formamide reagent and about 60% of the salt buffer.

7. 1. A biotin-streptavidin cleavage composition comprising: about 10% to about 50% by volume of a formamide reagent comprising formamide and an optional buffer; A biotin-streptavidin cleavage composition comprising sodium chloride, sodium citrate, and a balance salt buffer containing a biocompatible surfactant.

8. 1. A method comprising: introducing library fragments into a flow cell, said library fragments being attached to a streptavidin-coated solid support; introducing a biotin-streptavidin cleavage composition into the flow cell, wherein the biotin-streptavidin cleavage composition comprises: about 10% to about 50% by volume of a formamide reagent; the balance being a salt buffer; allowing the biotin-streptavidin cleavage composition to incubate in the flow cell at a temperature ranging from about 60°C to about 70°C, thereby releasing at least some of the library fragments from the solid support and seeding them onto amplification primers on the surface of the flow cell.

9. 9. The method of claim 8, wherein the biotin-streptavidin cleavage composition is allowed to incubate in the flow cell for a time ranging from about 2 minutes to about 5 minutes.

10. at least some of the streptavidin-coated solid supports become bound to biotin capture sites on the surface of the flow cell; 10. The method of claim 8 or 9, wherein allowing the biotin-streptavidin cleavage composition to incubate in the flow cell also releases at least some of the bound streptavidin-coated solid supports from the biotin capture sites.

11. 11. The method of claim 10, further comprising rinsing the biotin-streptavidin cleavage composition from the flow cell after incubation, thereby removing the streptavidin-coated solid support and any unseeded library fragments.

12. The method of any one of claims 8 to 11, further comprising the step of preparing the biotin-streptavidin cleavage composition by mixing the formamide reagent with the salt buffer.

13. 13. The method of claim 12, wherein the formamide reagent comprises 100% formamide.

14. 14. The method of claim 12 or 13, wherein the salt buffer comprises about 0.75 M sodium chloride and about 75 mM sodium citrate in water.

15. 15. The method of any one of claims 12 to 14, wherein the salt buffer further comprises about 0.25% to about 1.5% by weight of a biocompatible surfactant.

16. A kit comprising: a streptavidin-coated solid support; an adaptor sequence having biotin attached to one end, such that the biotin becomes attached to the streptavidin-coated solid support; a sample fluid containing genomic sequences that are fragmented and become attached to said adapter sequences; 1. A biotin-streptavidin cleavage composition comprising: about 10% to about 50% by volume of a formamide reagent; and a salt buffer as the balance.

17. 17. The kit of claim 16, wherein the formamide reagent is 100% formamide.

18. 18. The kit of claim 16 or 17, wherein the salt buffer comprises about 0.75 M sodium chloride and about 75 mM sodium citrate in water.

19. The kit of any one of claims 16 to 18, wherein the salt buffer further comprises about 0.25% to about 1.5% by weight of a biocompatible surfactant.

20. 1. A method comprising: introducing desthiobiotinylated library fragments into a flow cell, wherein the desthiobiotinylated library fragments are attached to a streptavidin-coated solid support; introducing a cleavage composition into the flow cell, wherein the cleavage composition is at a temperature in the range of about 18°C ​​to about 30°C, and the cleavage composition comprises: Free biotin, a salt buffer; raising the temperature of the cleavage composition to about 60°C to about 70°C, thereby releasing at least some of the library fragments from the solid support and seeding them onto amplification primers on the surface of the flow cell.

21. 21. The method of claim 20, wherein the salt buffer comprises about 0.75 M salt to about 0.85 M salt in water.

22. 22. The method of claim 20 or 21, wherein the free biotin is present in the cleavage composition at a concentration ranging from about 2.5 μM to about 10 mM.

23. 23. The method of any one of claims 20 to 22, wherein the salt buffer comprises about 0.75 M sodium chloride and about 75 mM sodium citrate in water.

24. 1. A library preparation fluid comprising: A liquid carrier; and library preparation beads in said liquid carrier, wherein each library preparation bead comprises: a solid support; a transposome complex attached to the solid support, wherein the transposome complex comprises: a transposase enzyme; a double-stranded molecule bound to the transposase enzyme, a transferred strand comprising a 3' transposon end sequence, an adapter sequence, a cleavage site, and a 5' ligation end sequence, wherein the adapter sequence and the 5' ligation end sequence are adjacent to the cleavage site; a non-transferred strand comprising a 3' transposon end sequence; and a splint sequence hybridized to at least a portion of the adapter sequence and at least a portion of the 5' ligation end sequence so as to splint the cleavage site.

25. 25. The library preparation fluid of claim 24, wherein the cleavage site is selected from the group consisting of a chemically cleavable cleavage site, an enzymatically cleavable cleavage site, and a photocleavable cleavage site.

26. 1. A method comprising: introducing a plurality of prepared library preparation beads into a reaction vessel, each of the prepared library preparation beads comprising: a solid support; a plurality of bridge molecules attached to the solid support, each of the bridge molecules comprising: a double-stranded DNA fragment; transferred strands attached at their 5' ends to each strand of the double-stranded DNA fragment, each transferred strand comprising a 3' transposon end sequence, a first adapter sequence, a cleavage site, and a 5' ligation end sequence, the adapter sequence and the 5' ligation end sequence being adjacent to the cleavage site; a second adapter sequence attached at its 3' end to each strand of the double-stranded DNA fragment, respectively; a splint sequence hybridized to at least a portion of the first adaptor sequence and at least a portion of the 5' ligation end sequence so as to splint the cleavage site; exposing the prepared library preparation beads to a cleavage agent to remove the cleavage site, thereby leaving the plurality of bridge molecules attached to the solid support via the splint; and heating the flow cell to a temperature at which the splint and the double-stranded DNA fragments dissociate.

27. the reaction vessel is a flow cell, at least some of the plurality of library preparation beads become immobilized on a surface of the flow cell; 27. The method of claim 26, wherein the method further comprises removing non-immobilized library preparation beads from the flow cell before exposing the library preparation beads to the cleavage agent.

28. 27. The method of claim 26, wherein the cleavage site is a chemical cleavage site, and exposing the prepared library preparation beads to the cleavage agent comprises introducing a chemical cleavage agent into the reaction vessel.

29. 27. The method of claim 26, wherein the cleavage site is an enzymatic cleavage site and exposing the prepared library preparation beads to the cleavage agent comprises introducing an enzymatic cleavage agent into the reaction vessel.

30. 27. The method of claim 26, wherein the cleavage site is a photocleavable cleavage site, and exposing the prepared library preparation beads to the cleavage agent comprises irradiating the reaction vessel with light of a wavelength that activates cleavage.

Images