Improvements in nucleic acid sequencing

Abstract

The present invention provides a method for preparing a template for high-throughput nucleic acid sequencing, the method comprising: a) providing a solid support and a library preparation, wherein the solid support comprises a plurality of nucleic acid primers, which fix an adaptor nucleic acid sequence hybridized to one or more of the nucleic acid primers, and the library preparation has a nucleic acid concentration of 400 pM or less; c) allowing a single-stranded fragment of the template nucleic acid to bind to the nucleic acid primer, thereby fixing the single-stranded fragment on the solid support; and d) repeating steps b) and c) at least three additional times, i.e., multiple loading or multiple rounds of template hybridization to improve, for example, the occupancy rate of the nanopore on the flow cell.

Description

Technical Field

[0001] The present invention relates to the improvement of high-throughput nucleic acid sequencing methods, and in particular to the improvement of methods for preparing templates for high-throughput nucleic acid sequencing. Background Art

[0002] Nucleic acid sequencing methods have been known in the art for many years. Some such methods are based on continuous cycles of incorporating fluorescently labeled nucleic acid analogs. In such "synthesis sequencing" or "cycle sequencing" methods, after each nucleotide addition, the identity of the added base is determined by detecting the fluorescent label.

[0003] Specifically, US 5,302,509 describes a method for sequencing a polynucleotide template, which involves using a DNA polymerase or DNA ligase to perform multiple extension reactions to continuously incorporate polynucleotides of a label complementary to the template strand. In this type of "synthesis sequencing" reaction, a new polynucleotide chain that base pairs with the template strand is constructed in the 5' to 3' direction by continuously incorporating a single nucleotide complementary to the template strand. The substrate nucleoside triphosphates used in the sequencing reaction are labeled at the 3' position using different 3' labels, thereby allowing the identity of the incorporated nucleotides to be determined when adding consecutive nucleotides.

[0004] In order to maximize the throughput of nucleic acid sequencing reactions, it is advantageous to be able to sequence multiple template molecules in parallel. Parallel processing of multiple templates can be achieved by using nucleic acid array technology. These arrays are usually composed of a high-density matrix of polynucleotides fixed to a solid carrier material.

[0005] The art has described various methods for making arrays for fixing nucleotide assays. Of particular interest, WO 98 / 44151 and WO 00 / 18957 both describe methods for nucleic acid amplification that allow amplification products to be fixed on a solid support to form an array consisting of clusters or "colonies" formed by a plurality of identical fixed polynucleotide chains and a plurality of identical fixed complementary chains. Nucleic acid molecules present in the DNA colonies on the clustered arrays prepared according to these methods can provide templates for sequencing reactions, for example as described in WO 98 / 44152.

[0006] In current high-throughput next generation sequencing (NGS) methods, sequencing chemistry is typically performed in a flow cell, where nucleic acids and other reagents are introduced. In order to prepare template strands, source nucleic acids are fragmented and fragments are connected to adapter sequences. These adapter sequences are designed to hybridize with short primer sequences fixed to a solid support in a flow cell, and then the sequencing template is amplified to produce clusters of identical template strands, which are then sequenced. Ideally, these clusters are of similar size and spaced apart from other clusters to achieve accurate resolution when imaged. Further sequencing reactions are then performed on the flow cell, where the results are imaged and sequence reads are compared to obtain the final sequence of the template.

[0007] To maintain physical separation of different clusters, the flow cell may include patterned nanopores, for example, formed by photolithography; only the nanopores include fixed primer sequences, and thus each cluster will be formed within the nanopore. The nanopore patterning determines the appropriate cluster spacing and position. To maintain the uniqueness of each cluster, it is desirable that a single template strand initially hybridizes within a given nanopore; therefore, template DNA is typically introduced into the flow cell at low molar amounts (approximately 10-20 pM). In addition, amplification can be performed using exclusion amplification techniques, which allow for simultaneous inoculation of template strands and amplification in the nanopores, thereby promoting monoclonal clusters.

[0008] As NGS methods continue to improve, attempts have been made to increase the density and number of nanopores within the flow cell, allowing for even greater sequencing throughput. However, the inventors have found that low molar concentrations of template DNA may be used inefficiently when using higher density patterned flow cells; for example, resulting in a lower proportion of occupied nanopores. Low template seeding concentrations may result in elevated replication levels and lower available yields. In addition, many commonly used library preparation methods may not be suitable for modification to obtain increased template nucleic acid concentrations. In order to mitigate low sample concentrations, the inventors have proposed an improved method that utilizes a flow cell as a nucleic acid capture device to increase the effective available concentration of low concentration sequencing templates. Summary of the invention

[0009] According to one aspect of the present invention, there is provided a method for preparing a template for a nucleic acid sequencing reaction, the method comprising:

[0010] a) providing a solid support, wherein the solid support comprises a plurality of nucleic acid primers immobilized thereon;

[0011] b) contacting a nucleic acid library preparation with the solid support, wherein the library preparation comprises a plurality of single-stranded fragments of a template nucleic acid to be sequenced, the template nucleic acid fragments further comprising one or more adapter nucleic acid sequences hybridized to one or more of the nucleic acid primers, and the library preparation has a nucleic acid concentration of 400 pM or less;

[0012] c) allowing the single-stranded fragment of the template nucleic acid to bind to the nucleic acid primer, thereby immobilizing the single-stranded fragment on the solid support; and

[0013] d) repeating steps b) and c) at least three additional times;

[0014] Thereby a solid support having the single-stranded fragment of the template nucleic acid immobilized thereon is provided.

[0015] According to another aspect of the present invention, there is provided a method for preparing a template for a nucleic acid sequencing reaction, the method comprising:

[0016] a) providing a flow cell, the flow cell comprising a solid support having a plurality of nanopores formed thereon, each nanopore comprising a plurality of nucleic acid primers immobilized on the solid support, and the nanopores being formed in a patterned array having a pitch of 500 nm or less;

[0017] b) contacting a nucleic acid library preparation with the flow cell, the library preparation comprising a plurality of single-stranded fragments of a template nucleic acid to be sequenced, the template nucleic acid fragments further comprising one or more adapter nucleic acid sequences hybridized to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less;

[0018] c) allowing a single-stranded fragment of the template nucleic acid to bind to the nucleic acid primer, thereby immobilizing the single-stranded fragment in the nanopore; and

[0019] d) repeating steps b) and c) at least three additional times;

[0020] Thereby a flow cell is provided with single-stranded fragments of the template nucleic acid immobilized in the nanopore.

[0021] The inventors have determined that such methods address the drawbacks of using low concentration nucleic acid libraries by allowing multiple loading "pushes" of the library. Each such push improves the occupancy of the nanopore. Multiple rounds of template hybridization and capture at low DNA concentrations increase the effective DNA concentrations, bringing them into the necessary range. This can significantly reduce library preparation concentration requirements and reduce template waste due to system dead volume, etc.

[0022] The method may include repeating steps b) and c) at least four times, five times, six times, seven times, eight times, nine times or more. It is believed that effective nucleic acid concentration is given by multiplying the number of replicates by the original nucleic acid concentration. For example, four replicates of a 200pM sample give an effective concentration of 800pM. In a preferred embodiment of the present invention, effective nucleic acid concentration is at least 800pM, preferably at least 1000pM.

[0023] The library preparation may have a nucleic acid concentration of 350 pM or less, 300 pM or less, 250 pM or less, or 200 pM or less. The library preparation may have a nucleic acid concentration of at least 50 pM, at least 100 pM, or at least 150 pM.

[0024] The repetition of the contacting step can be performed using the same library preparation or a different library preparation. For example, the method can include washing unbound template nucleic acid fragments from the flow cell, and recovering and reintroducing the unbound fragments into the flow cell. However, in a preferred embodiment, the repeated contacting step utilizes a fresh sample drawn from the same initial library preparation.

[0025] The method may include denaturing a library preparation comprising double-stranded fragments of a template nucleic acid to be sequenced to obtain single-stranded fragments of the template nucleic acid to be sequenced. The denaturation step may be performed on a flow cell; for example, a double-stranded fragment library may be introduced into a flow cell and then denatured to produce a single-stranded fragment library that contacts the flow cell. In a preferred embodiment, denaturation may be performed prior to contacting the flow cell.

[0026] The method may further comprise amplifying the immobilized single-stranded fragment of the nucleic acid, thereby generating multiple copies of the fragment. In a preferred embodiment, the amplification is performed using exclusion amplification; for example, as described in WO2013 / 188582.

[0027] In a preferred embodiment of the present invention, the solid support is glass and includes a patterned nanopore array thereon. The nanopore array can be formed by photolithography. The spacing of the nanopore array (i.e., the center-to-center distance between the nanopores) is preferably less than 750nm, more preferably less than 500nm, more preferably less than 400nm, and most preferably 350nm or less. The flow cell may include a plurality of channels formed on the solid support, wherein each channel includes a portion of the patterned nanopore array. For example, the flow cell may have a type produced by Illumina, Inc (San Diego, USA), which is used with the NovaSeq6000 system. An illustrative method for preparing a solid support having a specific type of patterned nanopore array thereon and a flow cell comprising such a solid support is described in EP 2 961 524.

[0028] The method may further comprise sequencing the immobilized single-stranded fragments of the nucleic acid. DETAILED DESCRIPTION

[0029] In its various aspects, the present invention generally relates to improvements in methods of high-throughput nucleic acid sequencing, and in particular to improvements in methods of preparing templates for high-throughput nucleic acid sequencing.

[0030] When referring to the attachment of molecules (e.g., nucleic acids) to solid supports, the terms "fixed" and "attached" are used interchangeably herein, and unless otherwise expressly or indicated by context, the two terms are intended to cover direct or indirect, covalent or non-covalent attachment. In certain embodiments of the invention, covalent attachment may be preferred, but generally all that is required is that the molecules (e.g., nucleic acids) remain fixed or attached to the support under conditions in which the support is intended to be used (e.g., in applications requiring nucleic acid amplification and / or sequencing). When referring to the attachment of nucleic acids to other nucleic acids, the terms "fixed" and "hybridized" are used herein, and generally refer to hydrogen bonding between complementary nucleic acids.

[0031] As used herein, when used in reference to a collection of items, the term "each" is intended to identify a single item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur if explicitly disclosed or the context clearly dictates otherwise.

[0032] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate can be nonporous or porous. The substrate can optionally be capable of absorbing liquids (e.g., due to porosity), but will generally be rigid enough so that the substrate does not significantly expand when absorbing liquids and does not substantially shrink when the liquids are removed by drying. Nonporous solid supports are generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic acid, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, polypropylene ... TM, cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, fiber bundles, and polymers. Particularly useful materials are glass. Other suitable substrate materials may include polymeric materials, plastics, silicon, quartz (fused quartz), borofloat glass, silica, silica-based materials, carbon, metals, optical fibers or fiber bundles, sapphire or plastic materials, such as COC and epoxides. A specific material may be selected based on the properties desired for a particular use. For example, a material that is transparent to radiation of a desired wavelength may be used for analytical techniques that utilize radiation of a desired wavelength, such as one or more of the techniques described herein. Conversely, it may be desirable to select a material that does not pass radiation of a specific wavelength (e.g., opaque, absorptive, or reflective). This can be used to form a mask to be used during the manufacture of a structured substrate; or for chemical reactions or analytical detection performed using a structured substrate. Other properties of the material that can be utilized are inertness or reactivity to certain reagents used in downstream processes; or ease of handling or low cost during manufacturing processing. Additional examples of materials that can be used in the structured substrates or methods of the present disclosure are described in US Serial No. 13 / 661,524 and US Patent Application Publication No. 2012 / 0316086A1, each of which is incorporated herein by reference.

[0033] Certain embodiments of the present invention utilize a solid support consisting of a substrate or matrix (e.g., a glass slide, polymer beads, etc.), which has been "functionalized" such as by applying an intermediate material layer or coating containing reactive groups, which allows covalent attachment to biomolecules such as polynucleotides. Examples of such carriers include, but are not limited to, substrates such as glass. In such embodiments, biomolecules (e.g., polynucleotides) can be directly covalently attached to the intermediate material, but the intermediate material itself can be non-covalently attached to the substrate or matrix (e.g., a glass substrate). The term "covalently attached to a solid support" should be interpreted accordingly as encompassing this type of arrangement. Alternatively, substrates (such as glass) can be treated to allow direct covalent attachment of biomolecules; for example, glass can be treated with hydrochloric acid to expose the hydroxyl groups of the glass, and triester phosphites are used to attach nucleotides directly to the glass via covalent bonds between the hydroxyl groups of the glass and the phosphate groups of the nucleotides.

[0034] In various aspects of the invention, attachment may be achieved via a sulfur-containing nucleophile such as a phosphorothioate present at the 5' end of the polynucleotide chain.

[0035] As used herein, the term "nanopore" refers to a discrete concave feature in a solid support having a surface opening that is completely surrounded by one or more void regions of the surface. The hole may have any of a variety of shapes at the opening in its surface, including but not limited to circular, elliptical, square, polygonal, star-shaped (with any number of vertices), etc. The cross-section of the hole taken orthogonally to the surface may be curved, square, polygonal, hyperbolic, conical, angular, etc. In a preferred embodiment of the present invention, the array of nanopores includes a plurality of nanopores disposed on a solid support; the patterned nanopore array includes a repeated arrangement of nanopores such that the relative arrangement of the nanopores at a portion of the solid support is the same as the relative arrangement of the nanopores at at least one other portion of the solid support. The spacing of the nanopore array is the center-to-center distance between two adjacent nanopores.

[0036] As will be understood by the technician, double-stranded nucleic acid will generally be formed by two complementary polynucleotide chains, which are composed of deoxyribonucleotides connected by phosphodiester bonds, but may additionally include one or more ribonucleotides and / or non-nucleotide chemical moieties and / or non-naturally occurring nucleotides and / or non-naturally occurring backbone bonds. Specifically, double-stranded nucleic acid may include non-nucleotide chemical moieties, such as joints or intermoleculars at the 5' ends of one or two chains. As a non-limiting example, double-stranded nucleic acid may include methylated nucleotides, uracil bases, thiophosphate groups, and may also include peptide conjugates, etc. Such non-DNA or non-natural modifications may be included to give nucleic acid some desired characteristics, such as to achieve covalent attachment to a solid support, or to serve as intermoleculars to position the cleavage site to the optimal distance from a solid support. Single-stranded nucleic acid is composed of one such polynucleotide chain. In the case where a polynucleotide chain is only partially hybridized with a complementary chain, for example, a long polynucleotide chain hybridized with a short nucleotide primer may still be referred to as a single-stranded nucleic acid herein.

[0037] The template nucleic acid to be sequenced will include a "target" region that is desired to be sequenced completely or partially. The nature of the target region is not limited to the present invention. It may have a previously known or unknown sequence, and may, for example, be derived from a genomic DNA fragment, a cDNA, etc. The template nucleic acid molecule also includes, for example, non-target sequences at the 5' and 3' ends of one or both chains (if double-stranded), which flank the target region. If the template nucleic acid is formed by solid phase nucleic acid amplification, these non-target sequences may be derived from primers used for amplification reactions. Alternatively, non-target sequences may be connected to fragment the target sequence so that it is incorporated into the nucleic acid molecule.

[0038] In embodiments of the invention, double-stranded nucleic acids may be subjected to denaturing conditions to provide single-stranded nucleic acids. Suitable denaturing conditions will be apparent to the skilled artisan, with reference to standard molecular biology protocols (Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY; Current Protocols, Ausubel et al., eds.).

[0039] Denaturation (and subsequent reannealing of the cleaved strands) results in a partially or substantially single-stranded sequencing template. The sequencing reaction can then be initiated by hybridizing a sequencing primer to the single-stranded portion of the template. In embodiments of the invention, sequencing can be performed using a strand displacement polymerase.

[0040] In embodiments of the present invention, as used herein, the term "solid carrier" refers to the material to which the polynucleotide molecules are attached. Suitable solid carriers are commercially available, and will be apparent to the technician. The carrier can be manufactured by materials such as glass, ceramics, silicon dioxide and silicon. A carrier with a gold surface can also be used. The carrier generally includes a flat (planar) surface, or at least one structure, in which the polynucleotide to be interrogated is in roughly the same plane. Any carrier of suitable size can be used. Alternatively, the solid carrier can be non-planar, such as microbeads.

[0041] In embodiments of the present invention, the method of the present invention can be used to prepare a template for nucleic acid sequencing. Single-stranded nucleic acids that can be amplified to provide a clustered array of nucleic acid colonies produced by solid phase nucleic acid amplification. In this context, the term "solid phase amplification" refers to an amplification reaction similar to standard PCR, except that forward and / or reverse amplification primers are fixed (e.g., covalently attached) to a solid support at or near the 5' end. Therefore, the product of the PCR reaction is an extended chain obtained by the extension of an amplification primer, which is fixed to a solid support at or near the 5' end. Solid phase amplification itself can be performed, for example, using procedures similar to those described in WO 98 / 44151 and WO 00 / 18957.

[0042] As a first step in generating colonies by solid phase amplification, a mixture of forward and reverse amplification primers can be immobilized or "grafted" onto the surface of a suitable solid support. The grafting step will generally involve covalently attaching the primers to the support at or near the 5' end, leaving the 3' end free for primer extension.

[0043] Amplification primers are typically oligonucleotide molecules with the following structure:

[0044] Forward primer: AL-S1

[0045] Reverse primer: AL-S2

[0046] wherein A represents an optional moiety allowing attachment to a solid support, L represents an optional linker moiety, and S1 and S2 are polynucleotide sequences allowing amplification of a substrate nucleic acid molecule comprising a target region that needs to be (completely or partially) sequenced.

[0047] The mixture of primers grafted to the solid support will generally contain substantially equal amounts of forward and reverse primers.

[0048] Group A can be any part (comprising non-nucleotide chemical modification), and it can be attached (preferably covalently attached) to solid support.In various aspects of the present invention, group A can comprise the sulfur-containing nucleophile present at the 5 ' end of polynucleotide chain, such as thiophosphate.Alternatively, when using suitable chemical substance that joint or nucleic acid are directly attached to solid support, group A can be omitted.

[0049] L represents a linker or spacer which may be included but is not strictly necessary. The linker may be included to ensure that the cleavage site present in the immobilized polynucleotide molecule produced as a result of the amplification reaction is positioned at an optimal distance from the solid support, or the linker itself may contain the cleavage site.

[0050] The linker may be of formula (CH2) n A carbon-containing chain wherein "n" is from 1 to about 1500, such as less than about 1000, preferably less than 100, such as 2-50, specifically 5-25. However, a variety of other linkers may be used, the only limitation to their structure being that the linker is stable under the conditions under which the polynucleotide is subsequently intended to be used, such as under conditions for DNA amplification and sequencing.

[0051] Linkers consisting not only of carbon atoms may also be used. Such linkers may include polyethylene glycol (PEG)

[0052] The linker formed mainly by the carbon atom chain and PEG can be modified to contain functional groups that interrupt the chain. Examples of such groups include ketones, esters, amines, amides, ethers, thioethers, sulfoxides, sulfones. Olefins, alkynes, aromatic or heteroaromatic moieties or cyclic aliphatic moieties (e.g., cyclohexyl) can be used alone or in combination with the presence of such functional groups. A cyclohexyl or phenyl ring can be connected to PEG or (CH2) for example through its 1- and 4-positions. n chain.

[0053] As an alternative to the above-mentioned linkers, which are mainly based on a linear chain of saturated carbon atoms optionally interrupted by unsaturated carbon atoms or heteroatoms, other linkers based on nucleic acids or monosaccharide units (e.g., dextrose) can be envisioned. It is also within the scope of the present invention to utilize peptides as linkers.

[0054] In other embodiments, the joint may include one or more nucleotides. Such nucleotides may also be referred to as "spacer" nucleotides in this article. Typically, 1 to 20, more preferably 1 to 15 or 1 to 10, and more specifically 2, 3, 4, 5, 6, 7, 8, 9 or 10 spacer nucleotides may be included. Most preferably, the primer will include 10 spacer nucleotides. Poly-T spacers are preferably used, but other nucleotides and combinations thereof may be used. In a preferred embodiment, the primer may include 10T spacer nucleotides.

[0055] To carry out the primer grafting reaction, a mixture of amplification primers is applied to a solid support under conditions that allow reaction between part A (if present) and the support, or between the nucleic acid and the support. The solid support can be suitably functionalized to allow covalent attachment via part A. The result of the grafting reaction is a substantially uniform distribution of primers on at least a portion of the solid support. In the case where the solid support includes a nanopore, then in a preferred embodiment, the primer is limited by the position of the nanopore and is not present in the void region of the solid support.

[0056] Nucleic acid library preparation is usually contacted with flow cell in free solution.Then, amplification reaction can be carried out as described in WO98 / 44151 basically.In short, after attaching primer, solid support is contacted with template to be amplified under conditions that allow hybridization between template and fixed primer.Template is usually added in free solution under suitable hybridization conditions, which will be obvious to technicians.Usually, hybridization conditions are, for example, 5xSSC at 40°C.Solid phase amplification can then be carried out, and the first step of amplification is primer extension step, in which nucleotides are added to the 3' end of the fixed primer hybridized with the template to produce a fully extended complementary chain.Therefore, this complementary chain will include a sequence that can bind to the second primer molecule fixed on the solid support at its 3' end.Another round of amplification (similar to standard PCR reaction) leads to the formation of a cluster or colony of template molecules bound to a solid support.Other amplification programs can be used, and will be known to technicians.For example, amplification can be carried out isothermal amplification using a strand displacement polymerase; or it can be exclusion amplification, as described in WO 2013 / 188582.

[0057] Sequences S1 and S2 in the amplification primers can be specific to a particular target nucleic acid that is desired to be amplified, but in other embodiments, sequences S1 and S2 can be "universal" primer sequences that are capable of amplifying any target nucleic acid with a known or unknown sequence that has been modified to be amplified using universal primers.

[0058] Suitable nucleic acids to be amplified with universal primers can be prepared by adding known adapter sequences to the 5' and 3' ends of the target polynucleotide to be amplified, modifying the polynucleotides comprising the target region to be amplified (and sequenced). The target molecule itself can be any polynucleotide molecule (e.g., a random fragment of human genomic DNA) that is desired to be sequenced. The adapter sequences can amplify these molecules on a solid support to form clusters using forward and reverse primers having the above-mentioned universal structure, wherein sequences S1 and S2 are universal primer sequences.

[0059] Adaptors are generally short oligonucleotides that can be synthesized by conventional means. Adaptors can be attached to the 5' and 3' ends of the target nucleic acid fragment by a variety of means (e.g., subcloning, connection, etc.). More specifically, two different adaptor sequences are attached to the target nucleic acid molecule to be amplified so that one adaptor is attached to one end of the target nucleic acid molecule and the other adaptor is attached to the other end of the target nucleic acid molecule. The resulting construct comprising the target nucleic acid sequence flanked by the adaptor may be referred to as a "substrate nucleic acid construct" in this article. Before being modified with the adaptor sequence, the target polynucleotide may be advantageously graded by size.

[0060] The adapter contains a sequence that allows nucleic acid amplification using an amplification primer molecule fixed on a solid support. These sequences in the adapter may be referred to as "primer binding sequences" herein. In order to serve as a template for nucleic acid amplification, the single strand of the template construct must contain a sequence complementary to the sequence S1 in the forward amplification primer (so that the forward primer molecule can bind to and initiate the synthesis of a complementary chain) and a sequence corresponding to the sequence S2 in the reverse amplification primer molecule (so that the reverse primer molecule can bind to the complementary chain). The length of the sequence in the adapter that allows hybridization with the primer molecule is generally about 20-40 nucleotides, but the present invention is not limited to sequences of this length.

[0061] The exact identity of sequences S1 and S2 in the amplification primers, and thus the homologous sequences in the adaptors, is generally not important to the invention, as long as the primer molecules are able to interact with the amplification sequences to direct PCR amplification. The design criteria for PCR primers are generally well known to those of ordinary skill in the art.

[0062] Solid phase amplification by methods similar to WO 98 / 44151 or WO 00 / 18957 will result in a clustered array consisting of colonies of "bridged" amplification products. The two chains of the amplification product will be fixed to the solid support at or near the 5' end, and this attachment is derived from the initial attachment of the amplification primers. Typically, the amplification product in each colony will be derived from the amplification of a single template (target) molecule.

[0063] The modification required for realizing the subsequent cutting bridge amplification product can be advantageously included in one or two amplification primers. Such modification can be placed in any position in the amplification primer, provided that this does not affect the efficiency of amplification reaction in a substantial degree. Therefore, the modification that can be cracked can form a part of the joint region L or one or both of the sequence S1 or S2. By way of example, the amplification primer can be modified to especially include the recognition sequence of glycol bond, uracil nucleotide, ribonucleotide, methylated nucleotide, peptide joint, PCR limiter or restriction endonuclease. Because all nucleic acid molecules prepared by solid phase amplification will eventually contain the sequence derived from the amplification primer, any modification in the primer will be carried in the amplification product.

[0064] Alternative amplification methods may be used (eg, isothermal amplification; or exclusion amplification, as described in WO2013 / 188582).

[0065] The present invention may also include a sequencing step; or aspects of the present invention may also encompass a method for sequencing a nucleic acid template generated using the method of the present invention. Therefore, the present invention provides a method for nucleic acid sequencing, the method comprising providing a template for nucleic acid sequencing using the method described herein, and performing a nucleic acid sequencing reaction to determine the sequence on at least one region of the template.

[0066] Sequencing may be performed using any suitable "sequencing by synthesis" technique, in which nucleotides are added successively to free 3' hydroxyl groups, resulting in the synthesis of a polynucleotide chain in the 5' to 3' direction. The identity of the added nucleotide is preferably determined after each addition.

[0067] The starting point of the sequencing reaction can be provided by annealing the sequencing primer to the single-stranded region of the template. Thus, the present invention encompasses methods wherein the nucleic acid sequencing reaction comprises hybridizing the sequencing primer to the single-stranded fragment of the template nucleic acid immobilized in the nanopore provided in the above aspects of the present invention, sequentially incorporating one or more nucleotides into a polynucleotide chain complementary to the region of the template to be sequenced, thereby identifying the bases present in the one or more incorporated nucleotides, and thereby determining the sequence of the region of the template.

[0068] The preferred sequencing method that can be used in the present invention relies on the use of modified nucleotides containing 3' blocking groups that can serve as chain terminators. Once the modified nucleotides have been incorporated into the growing polynucleotide chain complementary to the template region to be sequenced, there is no free 3'-OH group that can be used to guide further sequence extension, so the polymerase cannot add additional nucleotides. Once the nature of the base incorporated into the growing chain has been determined, the 3' block can be removed to allow the addition of the next continuous nucleotide. By sorting the products derived from these modified nucleotides, the DNA sequence of the DNA template can be inferred. If each of the modified nucleotides has been attached with different tags known to correspond to specific bases, it is beneficial to distinguish the bases added at each incorporation step, then such reactions can be completed in a single experiment. Alternatively, a separate reaction containing each of the modified nucleotides can be performed separately.

[0069] The modified nucleotide may carry a label to facilitate its detection. Preferably, this is a fluorescent label. Each nucleotide type may carry a different fluorescent label. However, the detectable label need not be a fluorescent label. Any label that allows detection of the incorporated nucleotide may be used.

[0070] One method for detecting fluorescently labeled nucleotides includes using a laser with a wavelength specific for the labeled nucleotide, or using other suitable illumination sources. Fluorescence from the label on the nucleotide can be detected by a CCD camera or other suitable detection device.

[0071] The methods of the present invention are not limited to use with the sequencing methods outlined above, but can be used in conjunction with essentially any sequencing method that relies on the sequential incorporation of nucleotides into a polynucleotide chain. Suitable techniques include, for example, pyrophosphate sequencing. TM >, FISSEQ (fluorescence in situ sequencing), MPSS (massively parallel signature sequencing), and sequencing by ligation-based methods.

[0072] The target polynucleotide sequenced using the method of the present invention can be any polynucleotide that is desired to be sequenced. The target polynucleotide can have a known, unknown or partially known sequence, for example in a re-sequencing application. Using the template preparation method described in detail herein, a template can be prepared starting from essentially any double-stranded target polynucleotide of known, unknown or partially known sequence. In the case of using an array, multiple targets of the same or different sequences can be sequenced in parallel. A particularly preferred application of the method is sequencing fragments of genomic DNA.

[0073] These and other aspects of the present invention will now be described with reference to the accompanying drawings, in which:

[0074] Figure 1 Shown is a schematic diagram of an illustrative flow cell that may be used with certain methods described herein.

[0075] Figure 2 The concept of the multihybridization workflow is shown.

[0076] Figure 3 Results obtained using 1 to 10 pushes (hybridization cycles) for various indicators are shown.

[0077] Figure 4 Library seeding efficiency was compared with two different multiple hybridization workflows.

[0078] Figure 5 Results from a 4-push workflow using a 200 pM library concentration are presented.

[0079] See also Figure 1 , which shows a schematic diagram of an illustrative flow cell that can be used with certain methods described herein. The flow cell is formed in three layers. The bottom layer 1 is formed of borosilicate glass and has a depth of 1000 μm. An etched silicon channel layer (with a depth of 100 μm) is placed on top to define 8 separate reaction channels. The top layer 3 (with a depth of 300 μm) includes two separate 8-hole series 4 and 4' aligned with the channels of the etched silicon channel layer to provide fluid communication with the contents of the channel when the flow cell is assembled in use. The borosilicate glass of the bottom layer 1 is etched with nanopores, which are arranged in a patterned array with a spacing of 350 nm and are arranged in line with the reaction channels. The void area between the channels is not etched and does not include nanopores. In use, the primer captured by the nucleic acid fragment template is bound to the nanopore.

[0080] In order to load the flow cell with the prepared sequencing library, the following protocol can be used. The initial library of double-stranded DNA for the sequencing technology to be used can be prepared in any suitable manner known in the art, comprising appropriate adapters. For example, a library preparation kit can be purchased from Illumina, Inc (San Diego, USA) to prepare a suitable library. The examples described herein are prepared using the TruSeqHuman Nano 450 preparation kit.

[0081] The following workflow simulates a 4-push multiple hybridization protocol. The total number of hybridization events can be varied up or down depending on the necessary use case and the desired final effective concentration.

[0082] Library denaturation and dilution:

[0083] 1. Dilute 20ul of double-stranded DNA library to the appropriate concentration using H2O or RSB buffer (available in the TruSeq Human Nano 450 preparation kit). To utilize the multiple hybridization workflow, this working concentration is 4 times lower than that required for the standard single event hybridization protocol.

[0084] 2. Mix the library with LDR denaturing reagent (100% formamide) at a 1:1 ratio.

[0085] 3. Heat to 65°C and incubate for 8 minutes to denature the double-stranded DNA template.

[0086] 4. Add 160ul HT1 (5X SSC + 0.1% Tween20) to dilute the denatured library to the final expected working concentration.

[0087] 5. Proceed to template hybridization

[0088] Template hybridization was performed using the Illumina NovaSeq6000 system using a multiplex hybridization workflow:

[0089] 1. Prime / wet the cartridge lines and flow cell with BB6 buffer.

[0090] 2. Heat the flow cell to 40°C.

[0091] 3. The denatured and diluted template is pumped to the flow cell with an initial flush factor large enough to completely cover the flow cell but without dilution by upstream BB6 buffer.

[0092] 4. Incubate for 5 minutes.

[0093] 5. Following incubation, an additional flow cell volume of denatured and diluted template was pulled to the flow cell and incubated for 5 minutes.

[0094] 6. Repeat step 5 two additional times, obtaining a total of 4 total hybridization events.

[0095] 7. Proceed to cluster generation.

[0096] Figure 2 The concept of multi-hybridization workflow is shown. A typical single push workflow is shown in the top line, where template hybridization is performed once, followed by cluster generation. If the template is loaded at a concentration of 800pM, the effective concentration remains 800pM. The second and third lines illustrate alternative ways to achieve the same concentration; two hybridization cycles (second line) using 400pM or four hybridization cycles (third line) of 200pM. It can be seen that multiple rounds of template hybridization / capture at low DNA concentrations increase the effective DNA concentration, thereby bringing them into the necessary range. This then significantly reduces library preparation concentration requirements and reduces template waste due to system dead volume, fluid lines, etc., and will potentially be able to run the rearrangement of low-yield library preparation.

[0097] Using the method described above, an initial 2 nM library was prepared and diluted to a 200 pM concentration.

[0098]

[0099] Typical library yields for different applications are shown below:

[0100]

[0101] Figure 3 Shown are the results obtained using 1 to 10 pushes (hybridization cycles) for various indices, wherein each workflow is designed to provide an 800pM effective concentration. The measured indices include cluster formation %, occupied nanopore %, remaining replication %, and available yield %. It can be seen that no matter how the number of pushes, the index is still in a small band, and multiple pushes of up to 10 times provide similar indicators to a single push of a higher concentration. The best for available yield and cluster formation is within the range of 4-6 pushes. The flow cell used has a 350nm nanopore spacing.

[0102] Figure 4 The library seeding efficiency was compared with two different multiple hybridization workflows (2 pushes vs. 4 pushes). An initial known amount of library was hybridized to the flow cell in one or more rounds, and the hybridized template was then eluted and quantified by qPCR. The initial input and uncaptured template fractions can be quantified using the hybridization fraction. The figure shows that 2 pushes and 4 pushes provide similar numbers of ssDNA molecules per nanopore, which increase with increasing total DNA exposure up to 700pM. As long as the final DNA exposure is maintained, the total number of pushes can be changed to obtain similar results. Relative to the 4 push protocol, the 2 push protocol utilizes 2xDNA concentration, but maintains similar final molecules per nanopore.

[0103] Figure 5 Results from a 4-push workflow are presented, using 200 pM library concentration (800 pM effective concentration), using libraries prepared from the TruSeq Human Nano 450 kit, performed on a 350 nm spacing flow cell. As the total DNA concentration increases to 800 pM, it can be seen that the % of nanowells occupied increases, and the % of replication decreases. It can be seen that the % passing the filter remains consistent as the concentration increases to 800 pM, showing that maintaining a lower concentration over multiple pushes results in optimal seeding.

[0104] The multiple hybridization workflow thus appears to be an effective approach to efficiently load high-density nanopore flow cells without excessively increasing replicated clusters, and demonstrates the use of sequencing flow cells as DNA capture devices to increase the effective concentration of samples to be sequenced, particularly when the sample preparation has a relatively low concentration yield. The preferred four-fold hybridization scheme reduces the library input concentration requirement by four-fold and potentially enables the running of rearrangements of low-yield library preparations.

Claims

1. A method for preparing a template for a nucleic acid sequencing reaction, the method comprising: a) providing a solid support, wherein the solid support comprises a plurality of nucleic acid primers immobilized thereon; b) contacting a nucleic acid library preparation with the solid support, wherein the library preparation comprises a plurality of single-stranded fragments of a template nucleic acid to be sequenced, the plurality of single-stranded fragments of the template nucleic acid further comprising one or more adapter nucleic acid sequences hybridized to one or more of the nucleic acid primers, and the library preparation has a nucleic acid concentration of 400 pM or less; c) allowing the single-stranded fragment of the template nucleic acid to bind to the nucleic acid primer, thereby immobilizing the single-stranded fragment on the solid support; and d) Repeat steps b) and c) at least three additional times; thereby providing a solid support having the single-stranded fragment of the template nucleic acid immobilized thereon; wherein the method does not include an amplification step until step d) is completed; wherein the repetition of the contacting step is performed using the same library preparation as used in step b).

2. The method according to claim 1, wherein the method is a method for preparing a template for a nucleic acid sequencing reaction, the method comprising: a) providing a flow cell, the flow cell comprising a solid support having a plurality of nanopores formed thereon, each nanopore comprising a plurality of nucleic acid primers immobilized on the solid support, and the nanopores being formed in a patterned array with a pitch of 500 nm or less, wherein the pitch is a center-to-center distance between two adjacent nanopores; b) contacting a nucleic acid library preparation with the flow cell, wherein the library preparation comprises a plurality of single-stranded fragments of a template nucleic acid to be sequenced, the plurality of single-stranded fragments of the template nucleic acid further comprising one or more adapter nucleic acid sequences hybridized to one or more of the nucleic acid primers, and the library preparation has a nucleic acid concentration of 400 pM or less; c) allowing a single-stranded fragment of the template nucleic acid to bind to the nucleic acid primer, thereby fixing the single-stranded fragment in the nanopore; as well as d) Repeat steps b) and c) at least three additional times; Thereby a flow cell is provided with single-stranded fragments of the template nucleic acid immobilized in the nanopore.

3. The method of claim 1 or claim 2, wherein the effective nucleic acid concentration calculated by multiplying the number of cycles in step c) by the nucleic acid concentration of the library preparation is at least 800 pM.

4. The method of claim 1 or claim 2, wherein the library preparation has a nucleic acid concentration of 250 pM or less.

5. The method according to claim 1 or claim 2, comprising denaturing the library preparation comprising double-stranded fragments of the template nucleic acid to be sequenced to obtain single-stranded fragments of the template nucleic acid to be sequenced used in step b).

6. The method of claim 1 or claim 2, further comprising amplifying the immobilized single-stranded fragment of the template nucleic acid, thereby generating multiple copies of the single-stranded fragment.

7. The method of claim 1 or claim 2, wherein the solid support is glass.

8. The method according to claim 1, wherein the solid support is located on a flow cell, wherein the solid support has a plurality of nanopores formed thereon, each nanopore comprises a plurality of nucleic acid primers immobilized on the solid support, and the nanopores are formed in the form of a patterned array. 9 . The method of claim 8 , wherein the nanopores are formed in a patterned array with a pitch of 500 nm or less, wherein the pitch is a center-to-center distance between two adjacent nanopores.

10. The method of claim 2, wherein the pitch of the nanohole array is 350 nm or less.

11. The method of claim 1, wherein the solid support is a microbead.

12. The method of claim 1 or claim 2, further comprising sequencing the immobilized single-stranded fragments of the template nucleic acid.

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