Method for improving clonality priority of polynucleotide clusters

Abstract

The present invention relates to compositions and methods for improving the generation of monoclonal clusters in arrays by adjusting the degree of homology between target nucleic acid adaptors and primers attached to the array to encode dynamic delays into the seeded target nucleic acids.

Description

[0001] priority

[0002] This application claims priority to U.S. Provisional Application No. 62 / 782,279, filed on December 19, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates particularly to generating amplicon sequencing clusters using exclusion amplification of target nucleic acids; and more particularly to increasing the number of monoclonal clusters. Background Art

[0004] Improvements in next-generation sequencing (NGS) technology have greatly increased sequencing speed and data output, resulting in huge sample throughput for current sequencing platforms. About 10 years ago, Illumina Genome Analyzers were capable of generating up to 1 gigabyte of sequence data per run. Today, Illumina NovaSeq TM The series of systems can generate up to 2 terabytes of data in two days, which represents a more than 2,000-fold increase in capacity.

[0005] One aspect of achieving this increased capacity is cluster generation. Cluster generation can include the creation of a library where the members of the library include a universal sequence present at each end. The library is loaded into a flow cell and individual members of the library are captured on a surface-bound oligomer complex complementary to the universal sequence. The individual members are then amplified into distinct clonal clusters by bridge amplification. After cluster generation is complete, individual clusters may contain approximately 1000 copies of a single member of the library, and the library is ready for sequencing.

[0006] One method of bridge amplification is exclusion amplification (ExAmp), also known as kinetic exclusion amplification. This method is a recombinase-promoted amplification reaction that utilizes a patterned array and isothermal conditions to amplify the library, thereby amplifying faster and using less reagents to generate clonal clusters in the wells of the array. The ExAmp method has been shown to be very useful for clonal cluster generation; however, conditions that result in more occupied wells also cause more polyclonal wells to be generated. Summary of the invention

[0007] Next-generation sequencing (NGS) technology relies on highly parallel sequencing of monoclonal populations of amplicons generated from a single target nucleic acid. Sequencing monoclonal populations of amplicons produces much higher signal-to-noise ratios, increased intensity, and an increased percentage of clusters passing filters, all of which contribute to improved data output and data quality.

[0008] The exclusion amplification method allows a single target nucleic acid to be amplified in each hole on the patterned flow cell, and a monoclonal population of amplicons is produced in the hole. Typically, the amplification rate of the first captured target nucleic acid in the hole is faster relative to the much slower target nucleic acid transport and capture rate in the hole. The first target nucleic acid captured in the hole can be rapidly amplified and fills the entire hole, thereby preventing other target nucleic acids from being captured in the same hole. Alternatively, if the second target nucleic acid is attached to the same hole after the first target nucleic acid, the rapid amplification of the first target nucleic acid usually fills enough holes to generate a signal through the filter. The use of exclusion amplification can also result in a super-Poisson distribution of monoclonal holes, i.e., the score of monoclonal holes in the array exceeds the score predicted by the Poisson distribution.

[0009] It is highly desirable to increase the super-Poisson distribution of useful clusters because more monoclonal wells result in more data output; however, the seeding of target nucleic acids into wells generally follows a spatial Poisson distribution, where the trade-off for more occupied wells is more polyclonal wells. One way to obtain a higher super-Poisson distribution is to have the seeding occur rapidly, followed by a delay between the seeded target nucleic acids. The delay (called a "kinetic delay" because it is believed to be generated by the kinetics of the biochemical reaction) causes one seeded target nucleic acid to start earlier than the other seeded targets.

[0010] Exclusion amplification works as follows, i.e., utilizing a recombinase to promote the invasion of primers (such as primers attached to holes) into double-stranded DNA (such as target nucleic acid) when it finds a sequence match. In order to maximize amplification efficiency, the standard practice of exclusion amplification is to utilize the complete identity between the intrusion primer and the adapter sequence. The inventors have determined the way to encode the kinetic delay to the inoculated target nucleic acid by adjusting the degree of homology between the target nucleic acid adapter and the primer attached to the hole. By reducing the average homology between the intrusion primer and the adapter sequence, even if the average rate of amplification is reduced, the rate of the so-called monoclonality of the hole is surprisingly improved. Generally, as more mismatches are introduced, the amplification efficiency decreases. Unexpectedly, when a mixture of adapter sequences with both higher and lower amplification efficiencies is used, the mixture does not perform as well as the performance average of the individual components, which is between high efficiency and low efficiency, but is superior to all single types of adapter sequences in both intensity and clusters passing through the filter.

[0011] definition

[0012] Unless otherwise specified, the terms used in this article should be understood to have ordinary meanings in the relevant field. Several terms used in this article and their meanings are listed below.

[0013] As used herein, the term "amplicon" refers to the product of nucleic acid replication when used to refer to nucleic acid, wherein the product has a nucleotide sequence identical or complementary to at least a portion of the nucleotide sequence of the nucleic acid.Amplicon can be produced by using nucleic acid, such as target nucleic acid or its amplicon as any of the multiple amplification methods of template, including such as polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), connection extension or connection chain reaction.Amplicon can be a nucleic acid molecule with a single copy (such as polymerase extension product) of a specific nucleotide sequence or multiple copies (such as concatemeric (concatemeric) products of RCA) of a nucleotide sequence. The first amplicon of a target nucleic acid is generally a complementary copy. Subsequent amplicon is a copy created from the target nucleic acid or from the first amplicon after producing the first amplicon. Subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.

[0014] As used herein, the term "amplification site" refers to a site in or on an array that can produce one or more amplicons. An amplification site can be further configured to contain, retain or attach at least one amplicon produced at the site.

[0015] As used herein, the term "array" refers to a population of sites that can be distinguished from each other according to relative positions. Different molecules located at different sites of the array can be distinguished from each other according to the position of the site in the array. The individual sites of the array may include one or more molecules of a specific type. For example, the site may include a single target nucleic acid molecule with a specific sequence, or the site may contain several nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of the array may be different features located on the same substrate. Exemplary features include, but are not limited to, holes in the substrate, beads (or other particles) in or on the substrate, projections from the substrate, ridges on the substrate, or channels in the substrate. The sites of the array may be separate substrates each with different molecules. Different molecules attached to separate substrates can be identified according to the position of the substrate on the surface associated with the substrate or according to the position of the substrate in a liquid or gel. Exemplary arrays where the separate substrate is located on the surface include, but are not limited to, those arrays with beads in the holes.

[0016] As used herein, the term "capacity" refers to the maximum amount of nucleic acid material that can occupy a site when used to refer to a site and a nucleic acid material, such as an amplicon derived from a target nucleic acid. For example, the term can refer to the total number of nucleic acid molecules that can occupy the site under specific conditions. Other measurements can also be used, including, for example, the total mass of nucleic acid material or the total number of copies of a specific nucleotide sequence that can occupy the site under specific conditions. Typically, the capacity of a site of a target nucleic acid will be substantially equivalent to the capacity of a site of an amplicon of the target nucleic acid.

[0017] As used herein, the term "capture agent" refers to a material, chemical substance, molecule, or portion thereof that can attach, retain, or bind to a target molecule (e.g., a target nucleic acid). Exemplary capture agents include, but are not limited to, capture nucleic acids complementary to at least a portion of a modified target nucleic acid (e.g., a universal capture binding sequence), members of a receptor-ligand binding pair that can bind to a modified target nucleic acid (or a linking portion attached thereto) (e.g., avidin, streptavidin, biotin, lectin, carbohydrates, nucleic acid binding proteins, epitopes, antibodies, etc.), or chemical reagents that can form a covalent bond with a modified target nucleic acid (or a linking portion attached thereto). In one embodiment, the capture agent is a nucleic acid. Nucleic acid capture agents can also be used as amplification primers.

[0018] When referring to nucleic acid capture agents, the terms "P5" and "P7" can be used. The terms "P5'" (P5 prime) and "P7'" (P7 prime) refer to the complements of P5 and P7, respectively. It should be understood that any suitable nucleic acid capture agent can be used in the methods proposed herein, and the use of P5 and P7 is only an exemplary embodiment. As disclosed in WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151 and WO 2000 / 018957, the use of nucleic acid capture agents such as P5 and P7 on flow cells is known in the art. Those skilled in the art will recognize that nucleic acid capture agents can also function as amplification primers. For example, any suitable nucleic acid capture agent can function as a forward amplification primer, whether immobilized or in solution, and can be used in the methods proposed herein to hybridize with a sequence (e.g., a universal capture binding sequence) and amplify the sequence. Similarly, any suitable nucleic acid capture agent can act as a reverse amplification primer, whether immobilized or in solution, and can be used in the methods proposed herein to hybridize with sequences (e.g., universal capture binding sequences) and amplify sequences. Given the available common sense and the teachings of this disclosure, those skilled in the art will understand how to design and use sequences suitable for capturing and amplifying target nucleic acids as proposed herein.

[0019] As used herein, the term "universal sequence" refers to a sequence region common to two or more target nucleic acids, wherein the molecule also has a sequence region different from each other. Universal sequences present in different members of a molecular set can allow the use of a capture nucleic acid population complementary to a portion of a universal sequence (e.g., a universal capture binding sequence) to capture multiple different nucleic acids. Non-limiting examples of universal capture binding sequences include sequences identical or complementary to P5 and P7 primers. Other non-limiting examples of universal capture binding sequences described in detail herein include sequences with reduced identity (e.g., one or more mismatches) or reduced complementarity with P5 and P7 primers, and / or sequences with a length less than P5 and P7 primers. Similarly, universal sequences present in different members of a molecular set can allow the use of a universal primer population complementary to a portion of a universal sequence, such as a universal primer binding site, to replicate or amplify multiple different nucleic acids. As described herein, target nucleic acid molecules can be modified to attach universal adapters (also referred to herein as adapters) at one or both ends of, for example, different target sequences.

[0020] As used herein, the term "adapter" and its derivatives, such as universal adaptors, generally refer to any linear oligonucleotide that can be connected to a target nucleic acid. In some embodiments, the adaptor is substantially non-complementary to the 3' end or 5' end of any target sequence present in the sample. In some embodiments, the suitable adaptor length is in the range of about 10-100 nucleotides, about 12-60 nucleotides, and about 15-50 nucleotides. Generally, the adaptor can include any combination of nucleotides and / or nucleic acids. In some aspects, the adaptor may include one or more cleavable groups at one or more positions. On the other hand, the adaptor may include a sequence that is substantially identical or substantially complementary to at least a portion of a primer, such as a capture nucleic acid. In some embodiments, the adaptor may include a barcode, also referred to as an index or tag, to assist downstream correction, identification, or sequencing. The terms "adapter" and "adapter" are used interchangeably.

[0021] As defined herein, "sample" and its derivatives are used in their broadest sense, and include any sample, culture, etc. suspected of including a target nucleic acid. In some embodiments, the sample comprises a chimeric or heterozygous form of DNA, RNA, PNA, LNA, nucleic acid. The sample may include any biological, clinical, surgical, agricultural, atmospheric or aquatic sample containing one or more nucleic acids. The term also includes any isolated nucleic acid sample, such as genomic DNA, fresh frozen or formalin-fixed paraffin-embedded nucleic acid specimens. Also contemplated are samples that may be from a single individual, a collection of nucleic acid samples from genetically related members, nucleic acid samples from genetically unrelated members, nucleic acid samples from a single individual (matching), such as tumor samples and normal tissue samples, or from two unique forms containing genetic material, such as samples from a single source of maternal and fetal DNA obtained from a maternal subject, or the presence of contaminating bacterial DNA in a sample containing plant or animal DNA. In some embodiments, the source of nucleic acid material may include nucleic acid obtained from a newborn, such as nucleic acid commonly used for newborn screening.

[0022] As used herein, the term "clone population" refers to a nucleic acid population that is homogeneous with respect to a particular nucleotide sequence. Homologous sequences are usually at least 10 nucleotides long, but can even be longer, including, for example, at least 50, at least 100, at least 250, at least 500 or at least 1000 nucleotides long. A clonal population can be derived from a single target nucleic acid. Typically, all nucleic acids in a clonal population will have the same nucleotide sequence. It should be understood that, without departing from clonality, a minority of mutations (e.g., due to amplification artifacts) can occur in a clonal population. It should also be understood that, without departing from clonality, a minority of different target nucleic acids (e.g., due to target nucleic acids that are not amplified or amplified to a limited extent) can occur in a clonal population.

[0023] As used herein, the term "different" when used to refer to nucleic acids means that the nucleic acids have nucleotide sequences that are different from each other. Two or more nucleic acids can have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different along a substantial portion of their length. For example, two or more nucleic acids can have target nucleotide sequence portions that are different from each other while also having common sequence regions that are identical to each other.

[0024] As used herein, the term "fluidic access," when used to refer to molecules in a fluid and sites in contact with a fluid, refers to the ability of molecules to move in or through a fluid to contact or enter the site. The term may also refer to the ability of molecules to separate from a site or leave a site to enter a solution. Fluidic access can occur when there are no obstacles that prevent molecules from entering a site, contacting a site, separating from a site, and / or leaving a site. However, as long as access is not absolutely prevented, even if diffusion is delayed, reduced, or altered, it is understood that a fluidic access exists.

[0025] As used herein, the term "double-stranded" when used to refer to a nucleic acid molecule refers to a nucleic acid molecule in which substantially all of the nucleotides are hydrogen bonded to complementary nucleotides. A partially double-stranded nucleic acid may have at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of its nucleotides hydrogen bonded to complementary nucleotides.

[0026] As used herein, the term "each" when used in reference to a collection of items is intended to identify individual items in the collection, but not necessarily every item in the collection unless the context clearly indicates otherwise.

[0027] As used herein, the term "excluded volume" refers to the volume of space occupied by a particular molecule to the exclusion of other such molecules.

[0028] As used herein, the term "gap region" refers to an area in a substrate or on a surface that is separated from other areas of the substrate or surface. For example, a gap region can separate one feature of an array from another feature of the array. Two regions separated from each other can be discrete, lacking contact with each other. In another example, a gap region can separate a first portion of a feature from a second portion of a feature. The separation provided by the gap region can be partial separation or complete separation. The gap region will typically have a surface material different from the surface material of the features on the surface. For example, the features of the array may have an amount or concentration of a capture agent that exceeds the amount or concentration present in the gap region. In some embodiments, the capture agent may not be present at the gap region.

[0029] As used herein, the term "polymerase" is intended to be consistent with its use in the art, and includes, for example, an enzyme that uses nucleic acid as a template strand to produce complementary repetitive nucleic acid molecules. Typically, DNA polymerase binds to the template strand, then moves downward along the template strand, thereby sequentially adding nucleotides to free hydroxyls at the 3' end of the growing strand of nucleic acid. DNA polymerases typically synthesize complementary DNA molecules from DNA templates, while RNA polymerases typically synthesize RNA molecules (transcription) from DNA templates. Polymerases can start chain growth using short RNA or DNA strands called primers. Some polymerases can displace the strand upstream of the site, where they are adding bases to the strand. Such polymerases are considered to be strand-displaced, meaning that they have the activity of removing complementary strands from the template strands read by the polymerase. Exemplary polymerases with strand-displacement activity include, but are not limited to, large fragments of Bsu (Bacillus subtilis), Bst (Bacillus stearothermophilus) polymerases, exo-Klenow polymerases, or sequencing-grade T7 exo-polymerases. Some polymerases degrade the strand ahead of them, effectively replacing it with the growing strand behind them (5' exonuclease activity). Some polymerases have activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified by mutation or other means to reduce or eliminate 3' and / or 5' exonuclease activity.

[0030] As used herein, the term "nucleic acid" is intended to be consistent with its use in the art, and includes naturally occurring nucleic acids and functional analogs thereof. Particularly useful functional analogs can hybridize with nucleic acids in a sequence-specific manner or can be used as a template for replicating a specific nucleotide sequence. Naturally occurring nucleic acids generally have a backbone comprising a phosphodiester bond. The analog structure can have an alternating backbone connection, including any of a variety of backbone connections known in the art. Naturally occurring nucleic acids generally have deoxyribose (e.g., found in deoxyribonucleic acid (DNA)) or ribose (e.g., found in ribonucleic acid (RNA)). Nucleic acids can contain any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural bases. In this regard, natural deoxyribonucleic acids can have one or more bases selected from adenine, thymine, cytosine or guanine, and ribonucleic acids can have one or more bases selected from uracil, adenine, cytosine or guanine. Useful non-natural bases that can be included in nucleic acids are known in the art. The term "target" when used to refer to a nucleic acid is intended in the context of the methods or compositions described herein as a semantic identifier for the nucleic acid and does not necessarily limit the structure or function of the nucleic acid beyond that expressly indicated in other contexts. A target nucleic acid having a universal sequence at each end, such as a universal adapter at each end, can be referred to as a modified target nucleic acid.

[0031] As used herein, the terms "recombinase loading protein" and "recombinase" are used interchangeably and are intended to be consistent with their use in the art, including, for example, RecA protein, T4UvsX protein, RB69 phage UvsX protein, any homologous protein or protein complex from any gate, or their functional variants. Eukaryotic RecA homologs are usually named Rad51 after the first member of the group is identified. Other non-homologous recombinases can be used instead of RecA, such as RecT or RecO.

[0032] As used herein, the term "single-stranded binding protein", also known as "SSB protein" or "SSB", is intended to refer to any protein that has the function of binding to single-stranded nucleic acids, such as to prevent premature annealing, protect single-stranded nucleic acids from nuclease digestion, remove secondary structure from nucleic acids, or promote the replication of nucleic acids. The term is intended to include, but is not limited to, proteins that are officially recognized as single-stranded binding proteins by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Exemplary single-stranded binding proteins include, but are not limited to, Escherichia coli SSB, T4 gp32, T7 gene 2.5SSB, bacteriophage phi 29SSB, RB69 bacteriophage gp32 protein, any homologous protein or protein complex from any phylum, and their functional variants.

[0033] As used herein, the term "accessory protein" is intended to refer to any protein that has the function of interacting with the recombinase and the single-stranded binding protein to help produce the nucleation of UvsX filaments on ssDNA. The terms "accessory protein", "recombinase accessory protein" and "recombinase accessory protein" are used interchangeably. Exemplary accessory proteins include, but are not limited to, T4 UvsY, RB69 bacteriophage UvsY protein, E. coli RecO, E. coli RecR, any homologous protein or protein complex from any phylum, and functional variants thereof.

[0034] As used herein, the term "transport" refers to the movement of molecules through a fluid. The term may include passive transport, such as the movement of molecules along their concentration gradient (e.g., passive diffusion). The term may also include active transport, whereby molecules can move along or against the concentration gradient of the molecule. Therefore, transport may include applying energy to move one or more molecules in a desired direction or to a desired position, such as an amplification site.

[0035] As used herein, the term "rate" is intended to be consistent with its meaning in chemical kinetics and biochemical kinetics when used to refer to transport, amplification, capture or other chemical processes. The rates of two processes can be compared with respect to maximum rate (e.g., when saturated), rate before steady state (e.g., before equilibrium), kinetic rate constant or other metrics known in the art. In a specific embodiment, the rate of a particular process can be determined with respect to the total time to complete the process. For example, the amplification rate can be determined with respect to the time spent to complete the amplification. However, it is not necessary to determine the rate of a particular process with respect to the total time to complete the process.

[0036] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0037] The words "preferred" and "preferably" refer to embodiments of the present invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not mean that other embodiments are not applicable, and is not intended to exclude other embodiments from the scope of the present invention.

[0038] The terms "include" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0039] It should be understood that wherever an embodiment is described with the language "comprising" or the like, otherwise similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0040] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0041] Conditions that are "suitable" for an event to occur, such as hybridization of two nucleic acid sequences, or "suitable" conditions are conditions that do not prevent such an event from occurring. Thus, these conditions allow, enhance, promote and / or facilitate the event.

[0042] As used herein, "providing" in the context of a composition, article, or nucleic acid means preparing the composition, article, or nucleic acid, purchasing the composition, article, or nucleic acid, or otherwise obtaining the compound, composition, article, nucleic acid.

[0043] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0044] References throughout the specification to "one embodiment," "embodiment," "certain embodiments," or "some embodiments" etc. mean that at least one embodiment of the present disclosure includes a particular feature, configuration, composition, or characteristic described in conjunction with the embodiment. Thus, the appearance of these phrases in various places throughout the specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, in one or more embodiments, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner.

[0045] For any method disclosed herein comprising discrete steps, the steps may be performed in any practicable order. Also, any combination of two or more steps may be performed simultaneously, where appropriate.

[0046] The above summary of the present invention is not intended to describe each disclosed embodiment or each implementation of the present invention. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided by example lists, which can be used in various combinations. In various cases, the enumerated lists serve only as representative groups and should not be interpreted as exclusive lists. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The following detailed description of exemplary embodiments of the present disclosure is best understood when read in conjunction with the following drawing figures.

[0048] Figure 1A and Figure 1B is a schematic diagram of an illustrative example of first and second capture sequences attached to a well of an array ( Figure 1A ), and a schematic diagram of an illustrative example of a target nucleic acid having a universal adaptor attached to each end ( Figure 1B ).

[0049] Figure 2 is a schematic diagram of an illustrative example of a hybridized single strand of a first capture nucleic acid and a target nucleic acid attached to a well of an array.

[0050] Figure 3A Displays the density of individual and grouped adapters passing through the filter. k / mm 2 , 1000 / mm2. The lanes refer to the lanes of the flow cell shown in Table 2 of the Examples. Figure 3B The ratios of final reads associated with individual mutant adapters are shown.

[0051] Figure 4A and Figure 4B Schematic diagram showing an illustrative example of strand invasion and replication ("RPA" refers to recombinase polymerase amplification). Figure 4AIn the example above, the recombinase promotes the invasion of the free P7 primer into a double-stranded template containing a homologous sequence (i.e., matching the P7 end). Perfect homology is not required (shown here by the introduction of two intentional mismatches in P7), but the rate of invasion and amplification can be reduced by adaptors of reduced homology (represented here by the smaller arrow of the mutant strand). Figure 4B In the present invention, recombinase-mediated invasion from either end occurs in the presence of an unmutated lawn-primer and effectively corrects mutations from the daughter strands, converting them back to perfect adapters. However, due to the reduced homology between the original strand and the lawn strand, the time delay until the first copy appears is proportional to the number and extent of mutations.

[0052] Figure 5A and Figure 5B Shown are the effects of short and mutant adapter libraries on amplification rates. Figure 5A In the process, one successful copy converts each template into a perfect template. However, the time constant for this transition depends on the degree of non-homology to be overcome (greater rates are indicated by thicker arrows). Figure 5B The slowed amplification rates in the short and mutant adapter libraries are indicated by the rightward shift of the real-time amplification curves.

[0053] Figure 6 Competition for clonal dominance between different templates on individual pads is shown. Inoculated templates with amplification preferences (i.e., kinetic delays) are shown; 1 = fastest, 6 = slowest. Equimolar ratios of templates are not necessary or even undesirable. A higher number of faster templates is preferred. However, even the slowest template (6) can fill the pad with monoclonal clusters when it has no competition on the pad.

[0054] The schematic diagrams are not necessarily drawn to scale. The same numbers used in the drawings refer to the same components, steps, etc. However, it should be understood that the use of numbers to refer to components in a given figure is not intended to limit the components labeled with the same numbers in another figure. In addition, the use of different numbers to refer to components is not intended to indicate that the components with different numbers cannot be the same or similar to the components with other numbers. DETAILED DESCRIPTION

[0055] Provided herein are compositions and methods related to increasing the generation of monoclonal clusters that can be used for sequencing.

[0056] The present disclosure provides a method for amplifying nucleic acids and a method for determining nucleic acid sequences. In one embodiment, the method includes providing an amplification reagent comprising (i) an array of amplification sites, and (ii) a solution having a plurality of different target nucleic acids. The amplification sites include at least two capture nucleic acid populations. One population, a first population, includes a first capture sequence, and the second population includes a second capture sequence. The different target nucleic acids include a first universal capture binding sequence at the 3' end. In one embodiment, the target nucleic acid is double-stranded. The first universal capture binding sequence has a lower affinity for the first capture sequence than a first universal capture binding sequence that is 100% complementary to the first capture sequence. For example, Figure 1A As shown, nucleic acids 100 of a first capture nucleic acid population comprise a first capture sequence 110 , wherein the nucleic acids 100 are attached to a surface of an amplification site 120 . Figure 1B Shown in is a double-stranded target nucleic acid 130 comprising a universal adaptor 140 at each end, and a first universal capture binding sequence 150 at the 3′ end of each universal adaptor 140 .

[0057] Optionally, the different target nucleic acids further comprise a second universal capture binding sequence at the 5' end. The complement of the second universal capture binding sequence has a lower affinity for the second capture sequence than the complement of the second universal capture binding sequence that has 100% complementarity with the second capture sequence. For example, Figure 1A As shown, nucleic acids 160 of the second capture nucleic acid population comprise a first capture sequence 170 , wherein the nucleic acids 160 are attached to the surface of the amplification site 120 . Figure 1B Shown in FIG. 1 is a double-stranded target nucleic acid 130 comprising a universal adaptor 140 at each end, and a second universal capture binding sequence 180 at the 5′ end of each universal adaptor 140 .

[0058] The method further includes reacting the amplification reagents to produce a plurality of amplification sites, each of which has a clonal population of amplicons of a respective target nucleic acid from the solution. The reaction includes transporting the different target nucleic acids to the amplification sites and amplifying the target nucleic acids at the amplification sites. For example, Figure 2 As shown, nucleic acids 200 of the first capture nucleic acid population include a first capture sequence 210, wherein the nucleic acids 200 are attached to the surface of an amplification site 220. One strand of a target nucleic acid 230 including a first universal capture binding sequence 250 at the 3' end of the single strand is hybridized to the first capture sequence 210 of the nucleic acid 200. The first universal capture binding sequence 250 includes an "X" to indicate the presence of a mismatch between the first universal capture binding sequence 250 and the first capture sequence 210. This can then be subjected to cluster amplification, such as by bridge amplification, to result in cluster generation.

[0059] Also provided herein is a method for producing a nucleic acid library. The library can be used for the amplification method described herein. The method includes providing a solution of multiple different target nucleic acids. In one embodiment, the target nucleic acid is double-stranded. Universal adapters are connected to both ends of the double-stranded target nucleic acid to form a first plurality of modified target nucleic acids, wherein each modified target nucleic acid comprises a target nucleic acid flanked by a universal adapter. The universal adapter comprises a double-stranded nucleic acid region and a single-stranded non-complementary nucleic acid chain region. The single-stranded non-complementary nucleic acid chain region comprises a first universal capture binding sequence at the 3' end. The first universal capture binding sequence has a smaller affinity for the first capture sequence than the first universal capture binding sequence having 100% complementarity with the first capture sequence. Optionally, the single-stranded non-complementary nucleic acid chain region comprises a second universal capture binding sequence at the 5' end. The complement of the second universal capture binding sequence has a smaller affinity for the second capture sequence than the second universal capture binding sequence having a complement of 100% complementarity with the second capture sequence.

[0060] Array

[0061] The array of amplification sites used in the methods described herein can exist as one or more substrates. Example types of substrate materials that can be used for arrays include glass, modified glass, functionalized glass, inorganic glass, microspheres (e.g., inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or fiber bundles, polymers, and porous (e.g., microtiter) plates. Exemplary plastics include copolymers of acrylic acid, polystyrene, styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon. TM Exemplary silica-based materials include silicon and various forms of modified silicon.

[0062] In specific embodiments, the substrate can be within or part of a container such as a well, tube, channel, dish, culture dish, bottle, etc. A particularly useful container is a flow cell, for example, as described in U.S. Patent No. 8,241,573 or Bentley et al., Nature 456:53-59 (2008). An exemplary flow cell is a flow cell available from Illumina, Inc. (San Diego, Calif.). Another particularly useful container is a well in a multiwell plate or microtiter plate.

[0063] In some embodiments, the site of the array can be configured as features on the surface. These features can exist in any of a variety of desired forms. For example, the site can be a hole, a pit, a channel, a ridge, a raised area, a nail, a column, etc. As described above, the site can contain a pearl. However, in a specific embodiment, the site does not need to contain a pearl or a particle. Exemplary sites include holes present in substrates for commercial sequencing platforms sold by 454 Life Sciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.). Other substrates with holes include, for example, etched optical fibers and other substrates described in U.S. Pat. No. 6,266,459; U.S. Pat. No. 6,355,431; U.S. Pat. No. 6,770,441; U.S. Pat. No. 6,859,570; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; U.S. Pat. No. 6,274,320; U.S. Pat. No. 8,262,900; U.S. Pat. No. 7,948,015; U.S. Patent Publication No. 2010 / 0137143; U.S. Pat. No. 8,349,167, or PCT Publication No. WO 00 / 63437. In several cases, substrates with beads in the holes are exemplified in these references. In the methods or compositions of the present disclosure, the substrates containing holes can be used with or without beads. In some embodiments, the wells of the substrate may contain a gel material (with or without beads), as described in US Pat. No. 9,512,422.

[0064] The sites of the array can be metal features on a non-metallic surface, such as glass, plastic, or other materials exemplified above. The metal layer can be deposited on the surface using methods known in the art, such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering, etc. Any of a variety of commercial instruments can be used appropriately, including, for example, , Ionfab or Optofab System (Oxford Instruments, UK). Metal layers can also be deposited by electron beam evaporation or sputtering, as described in Thornton, Ann. Rev. Mater. Sci. 7: 239-60 (1977). Metal layer deposition techniques, such as those exemplified above, can be combined with photolithography to create metal areas or patches on a surface. Exemplary methods for combining metal layer deposition techniques and photolithography are provided in U.S. Pat. No. 8,778,848 and U.S. Pat. No. 8,895,249.

[0065] The feature array can appear as a grid of spots or patches. The features can be positioned in a repeating pattern or an irregular non-repeating pattern. Particularly useful patterns are hexagonal patterns, rectilinear patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, etc. Asymmetric patterns can also be useful. The spacing between the nearest neighbor features of different pairs can be the same, or there can be variations in the spacing between the nearest neighbor features of different pairs. In a specific embodiment, the features of the array can each have a spacing greater than about 100 nm. 2 , 250nm 2 , 500nm 2 , 1μm 2 , 2.5μm 2 , 5μm 2 , 10μm 2 , 100μm 2 or 500μm 2 Alternatively, or in addition, the features of the array may each have an area of ​​less than about 1 mm 2 , 500μm 2 , 100μm 2 , 25μm 2 , 10μm 2 , 5μm 2 , 1μm 2 , 500nm 2 , or 100nm 2 In practice, the region may have a size within a range between an upper limit and a lower limit selected from the examples above.

[0066] For embodiments including arrays of features on the surface, the features can be discrete, separated by gap regions. There can be variations in the size of the features and / or the spacing between regions, so that the array can be high density, medium density or relatively low density. High density arrays are characterized by having regions with spacings less than about 15 μm. Medium density arrays have regions with spacings of about 15 to 30 μm, while low density arrays have regions with spacings greater than 30 μm. Arrays useful in the present disclosure can have regions with spacings less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm.

[0067] In a specific embodiment, the array may include a collection of beads or other particles. The particles may be suspended in a solution or may be positioned on the surface of a substrate. Examples of bead arrays in solution are those commercialized by Luminex (Austin, Tex.). Examples of arrays with beads positioned on the surface include arrays in which beads are positioned in holes, such as BeadChip arrays (Illumina Inc., San Diego, Calif.) or substrates for sequencing platforms from 454 Life Sciences (a subsidiary of Roche, Basel, Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.). Other arrays with beads on the surface are described in U.S. Pat. No. 6,266,459; U.S. Pat. No. 6,355,431; U.S. Pat. No. 6,770,441; U.S. Pat. No. 6,859,570; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; U.S. Pat. No. 6,274,320; US 2009 / 0026082 A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143 A1; US ​​2010 / 0282617 A1; or PCT Publication No. WO 00 / 63437. Several of the above references describe methods of attaching target nucleic acids to beads prior to loading the beads in or on an array substrate. However, it should be understood that the beads can be made to contain amplification primers, which can then be used to load the array, thereby forming amplification sites for the methods described herein. As previously described herein, substrates can be used without beads. For example, amplification primers can be directly attached to a well or gel material in a well. Thus, references illustrate materials, compositions or devices that can be modified for use in the methods and compositions described herein.

[0068] The amplification site of the array may include multiple capture agents that can bind to the target nucleic acid. In one embodiment, the capture agent includes capture nucleic acid. Under typical conditions for preparing an array for sequencing, the nucleotide sequence of the capture nucleic acid of the present disclosure is complementary to the sequence of one or more target nucleic acids. On the contrary, the nucleotide sequence of the capture nucleic acid of the present disclosure is not completely complementary to the sequence of one or more target nucleic acids. The nucleotide sequence of the capture nucleic acid that can be used in the method provided by the present disclosure is described in detail herein. In some embodiments, the capture nucleic acid can also serve as a primer for amplifying the target nucleic acid (regardless of whether it also includes a universal sequence). In some embodiments, a capture nucleic acid population includes a P5 primer or its complement, and a second capture nucleic acid population includes a P7 primer or its complement.

[0069] In a specific embodiment, a capture agent such as a capture nucleic acid can be attached to an amplification site. For example, a capture agent can be attached to the surface of an array feature. Attachment can be through an intermediate structure, such as a bead, a particle, or a gel. The example of attaching capture nucleic acids to an array through a gel is described in U.S. Patent No. 8,895,249, and further illustrated by a flow cell that can be purchased from Illumina Inc. (San Diego, Calif.) or described in WO 2008 / 093098. Exemplary gels that can be used in the methods and devices described herein include, but are not limited to, those with a colloidal structure, such as agarose; a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide, SFA (e.g., see U.S. Patent Publication No. 2011 / 0059865A1) or PAZAM (e.g., see U.S. Provisional Patent Application Serial No. 61 / 753,833 and U.S. Patent No. 9,012,022). Attachment via beads can be achieved as illustrated in the description and references cited previously herein.

[0070] In some embodiments, the features on the surface of the array substrate are discontinuous and separated by the gap regions on the surface. Compared with the features of the array, the gap regions with a substantially lower number or concentration of capture agents are advantageous. The gap regions lacking capture agents are particularly advantageous. For example, a relatively small amount or absence of capture parts at the gap regions is conducive to locating the target nucleic acid and the clusters subsequently produced to the desired features. In a specific embodiment, the feature can be a concave feature in a surface (e.g., a hole), and the feature can contain a gel material. The features containing gel can be separated from each other by the gap regions on the surface, and there is substantially no gel in the gap regions, or if there is, the gel can not support the positioning of nucleic acids substantially. The method and composition of preparing and using a substrate having a feature such as a hole containing gel are described in U.S. Patent No. 9,512,422.

[0071] Target nucleic acid

[0072] The solution of amplification reagent used in the methods described herein includes a target nucleic acid. The terms "target nucleic acid", "target fragment", "target nucleic acid fragment", "target molecule" and "target nucleic acid molecule" are used interchangeably to refer to nucleic acid molecules that are expected to be sequenced, such as sequenced on an array. The target nucleic acid can be substantially any nucleic acid of known or unknown sequence. For example, it can be a fragment of genomic DNA or cDNA. Sequencing can result in determining the sequence of all or part of the target molecule. The target can be derived from a primary nucleic acid sample that has been randomly fragmented. In one embodiment, the target can be processed into a template suitable for amplification by placing a universal amplification sequence at the end of each target fragment, such as a sequence present in a universal adapter.

[0073] The primary nucleic acid sample can be derived from a double-stranded DNA (dsDNA) form from the sample (e.g., genomic DNA fragments, PCR and amplification products, etc.) or can be derived from a sample (such as DNA or RNA) in a single-stranded form and converted to a dsDNA form. For example, mRNA molecules can be replicated into double-stranded cDNA suitable for the methods described herein using standard techniques well known in the art. The precise sequence of the polynucleotide molecules from the primary nucleic acid sample is generally not important to the present disclosure and may be known or unknown.

[0074] In one embodiment, the primary polynucleotide molecule from the primary nucleic acid sample is a DNA molecule. More particularly, the primary polynucleotide molecule represents the entire genetic complement of an organism, and is a genomic DNA molecule, which includes both introns and exon sequences, and non-coding regulatory sequences, such as promoters and enhancer sequences. In one embodiment, a specific subset of a polynucleotide sequence or genomic DNA can be used, such as a specific chromosome. More particularly, the sequence of the primary polynucleotide molecule is unknown. Still more particularly, the primary polynucleotide molecule is a human genomic DNA molecule. Can be before or after any random fragmentation process, and before or after the universal adapter sequence is connected, chemical or enzyme treatment DNA target fragments.

[0075] Nucleic acid samples can include high molecular weight materials, such as genomic DNA (gDNA). Samples can include low molecular weight materials, such as nucleic acid molecules obtained from FFPE or archived DNA samples. In another embodiment, low molecular weight materials include enzyme or mechanical fragmentation DNA. Samples can include cell-free circulating DNA. In some embodiments, samples can include nucleic acid molecules obtained from biopsy, tumor, scrapings, swabs, blood, mucus, urine, blood plasma, semen, hair, laser capture microdissection, surgical resection and other clinical or laboratory samples obtained. In some embodiments, samples can be epidemiological, agricultural, forensic or etiological samples. In some embodiments, samples can include nucleic acid molecules obtained from animals such as humans or mammalian sources. In another embodiment, samples can include nucleic acid molecules obtained from non-mammal sources such as plants, bacteria, viruses or fungi. In some embodiments, the source of nucleic acid molecules can be archived or exhausted samples or species.

[0076] In addition, the methods and compositions disclosed herein can be used to amplify nucleic acid samples with low-quality nucleic acid molecules, such as genomic DNA from degradation and / or fragmentation of forensic samples. In one embodiment, forensic samples may include nucleic acids obtained from crime scenes, nucleic acids obtained from missing persons DNA databases, nucleic acids obtained from laboratories associated with forensic investigations, or include forensic samples obtained from law enforcement agencies, one or more military departments, or any such personnel. Nucleic acid samples can be purified samples or lysates containing crude DNA, such as from oral swabs, paper, fabrics, or other substrates that can be impregnated with saliva, blood, or other body fluids. Therefore, in some embodiments, nucleic acid samples may include a small amount of DNA (such as genomic DNA) or a fragmented portion of the DNA. In some embodiments, the target sequence may be present in one or more body fluids, including but not limited to blood, sputum, plasma, semen, urine, and serum. In some embodiments, the target sequence may be obtained from hair, skin, tissue samples, autopsies, or remains of victims. In some embodiments, nucleic acids including one or more target sequences may be obtained from deceased animals or humans. In some embodiments, the target sequence may include nucleic acids obtained from non-human DNA, such as microorganisms, plants, or entomology DNA. In some embodiments, the target sequence or amplified target sequence is for the purpose of human identification. In some embodiments, the disclosure relates to methods for identifying characteristics of forensic samples. In some embodiments, the disclosure generally relates to methods for human identification using one or more target-specific primers disclosed herein or one or more target-specific primers designed using primer design criteria outlined herein. In one embodiment, a forensic or human identification sample comprising at least one target sequence can be amplified using any one or more target-specific primers disclosed herein or obtained using primer standards outlined herein.

[0077] Other non-limiting examples of biological sample sources may include complete organisms and samples obtained from patients. Biological samples can be obtained from any biological fluid or tissue, and can be in various forms, including liquid fluids and tissues, solid tissues and preservation forms, such as drying, freezing and fixed forms. Samples can be any biological tissue, cell or body fluid. Such samples include but are not limited to sputum, blood, serum, plasma, blood cells (such as leukocytes), ascites, urine, saliva, tears, sputum, vaginal fluid (discharge), cleaning fluids obtained during medical procedures (for example, pelvis or other cleanings obtained during biopsy, endoscopy or surgery), tissue, nipple aspirates, core or fine needle biopsy samples, body fluids containing cells, free floating nucleic acids, peritoneal fluid and pleural fluid, or cells from them. Biological samples can also include tissue sections, such as frozen sections or fixed sections taken for histological purposes, or microdissected cells or their extracellular parts. In some embodiments, sample can be a blood sample, such as for example a whole blood sample. In another example, sample is an untreated dried blood spot (DSS) sample. In another example, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In yet another example, the sample is a saliva sample. In yet another example, the sample is a dried saliva spot (DSS) sample.

[0078] Exemplary biological samples from which target nucleic acids can be derived include, for example, samples from eukaryotic organisms, such as mammals, e.g., rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates, humans, or non-human primates; plants, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola, or soybeans; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, bees, or spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi, such as Pneumocystis carinii; carinii), Takifugu rubripes, yeast, Saccharamoyces cerevisiae, or Schizosaccharomyces pombe; or Plasmodium falciparum. The target nucleic acid may also be derived from a prokaryotic organism, such as bacteria, Escherichia coli, Staphylococcus, or Mycoplasma pneumoniae; archaea; viruses, such as human hepatitis C virus or human immunodeficiency virus; or viroids. The target nucleic acid may be derived from a homogenous culture or population of the above organisms, or alternatively from a collection of several different organisms, such as in a community or ecosystem.

[0079] Random fragmentation refers to fragmenting polynucleotide molecules from primary nucleic acid samples in a disordered manner by enzymatic, chemical or mechanical means.Magnetic fragmentation methods are known in the art, and standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, 3rd edition) are used.In one embodiment, fragmentation can be achieved using a process commonly referred to as tag fragmentation (tagmentation).Tag fragmentation uses a transposome complex, and is combined into a one-step fragmentation and connection to add a universal adapter (Gunderson et al., WO 2016 / 130704).For the sake of clarity, the nucleic acid sequence of a larger fragment remains intact (i.e., retains a complete nucleic acid sequence), and such smaller fragments of a larger section of nucleic acid produced by amplifying smaller fragments by specific PCR are not equivalent to fragmenting a larger section of nucleic acid, because the larger section of nucleic acid sequence remains intact (i.e., not amplified by PCR).In addition, random fragmentation is designed to produce fragments, regardless of the sequence identity or position of the nucleotides containing and / or surrounding the fracture. More particularly, random fragmentation is by mechanical means, such as nebulization or sonication, to produce fragments of about 50 base pairs in length to about 1500 base pairs in length, still more particularly 50-700 base pairs in length, more particularly 50-400 base pairs in length. Most particularly, the method is used to produce smaller fragments of 50-150 base pairs in length.

[0080] By mechanical means (such as atomization, ultrasonic treatment and Hydroshear), polynucleotide molecule fragmentation is produced with a heterogeneous mixed fragment of flat end and 3'- and 5'-overhang. Therefore, it is desirable to repair the fragment ends using methods known in the art or test kits (such as Lucigen DNA terminator end repair kit) to produce the best ends for inserting into, for example, a flat site of a cloning vector. In a specific embodiment, the fragment ends of a nucleic acid colony are flat ends. More particularly, the fragment ends are flat ends and phosphorylated. The phosphate moiety can be introduced by enzyme treatment, which, for example, uses polynucleotide kinase.

[0081] The target nucleic acid colony or its amplification can have the average chain length that is expected or suitable for the particular application of the methods or compositions described herein.For example, the average chain length can be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides or 50 nucleotides.Or / in addition, the average chain length can be greater than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides or 100,000 nucleotides.The average chain length of the target nucleic acid colony or its amplicon can be in the range between the maximum value and the minimum value described above.It should be understood that the amplicon produced at the amplification site (or otherwise prepared or used in this article) can have the average chain length in the range between the upper limit and the lower limit selected from the above exemplification.

[0082] In some cases, the target nucleic acid colony can be produced under certain conditions or otherwise configured to have the maximum length of its members. For example, the maximum length of the member present in one or more steps of the methods described herein or in a specific composition can be less than 100,000 nucleotides, less than 50,000 nucleotides, less than 10,000 nucleotides, less than 5,000 nucleotides, less than 1,000 nucleotides, less than 500 nucleotides, less than 100 nucleotides or less than 50 nucleotides. Or / in addition, the target nucleic acid colony or its amplicon can be produced under certain conditions or otherwise configured to have the minimum length of its target member. For example, the minimum length of the member present in one or more steps of the methods described herein or in a specific composition can be greater than 10 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 100 nucleotides, greater than 500 nucleotides, greater than 1000 nucleotides, greater than 5,000 nucleotides, greater than 10,000 nucleotides, greater than 50,000 nucleotides or greater than 100,000 nucleotides. The maximum and minimum chain lengths of the target nucleic acid in the population can be in the range between the maximum and minimum values ​​set forth above. It should be understood that the amplicon produced at the amplification site (or otherwise prepared or used herein) can have a maximum and / or minimum chain length in the range between the upper and lower limits of the above exemplifiers.

[0083] In a specific embodiment, the size of the target nucleic acid is determined relative to the area of ​​the amplification site, for example, to promote exclusion amplification. For example, the area of ​​each site of the array may be greater than the diameter of the exclusion volume of the target nucleic acid to achieve exclusion amplification. Taking, for example, an embodiment using a feature array on a surface as an example, the area of ​​each feature may be greater than the diameter of the exclusion volume of the target nucleic acid transported to the amplification site. The exclusion volume of the target nucleic acid and its diameter can be determined, for example, by the length of the target nucleic acid. Methods for determining the exclusion volume of nucleic acids and the diameter of the exclusion volume are described in, for example, U.S. Patent No. 7,785,790; Rybenkov et al., Proc. Natl. Acad. Sci. USA 90: 5307-5311 (1993); Zimmerman et al., J. Mol. Biol. 222: 599-620 (1991); or Sobel et al., Biopolymers 31: 1559-1564 (1991).

[0084] In a specific embodiment, a target fragment sequence having a single protruding nucleotide is prepared by, for example, the activity of certain types of DNA polymerases such as Taq polymerase or Klenow exo minus polymerase, which has a non-template-dependent terminal transferase activity that adds a single deoxynucleotide, such as deoxyadenosine (A), to the 3' end of a DNA molecule (e.g., a PCR product). Such enzymes can be used to add a single nucleotide "A" to the blunt-ended 3' end of each chain of a double-stranded target fragment. Therefore, "A" can be added to the 3' end of each end-repaired chain of a double-stranded target fragment by reacting with Taq or Klenow exo minus polymerase, and the universal adapter polynucleotide construct can be a T construct having a compatible "T" protrusion present at the 3' end of each region of the double-stranded nucleic acid of the universal adapter. This terminal modification also prevents self-ligation of both the vector and the target, so that there is a preference for the formation of combined connected adapter-target-adapter molecules.

[0085] In some cases, before being used for the method or composition herein, the target nucleic acid derived from such source can be amplified. Any of a variety of known amplification techniques can be used, including but not limited to polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA) or random prime amplification (random prime amplification, RPA). It should be understood that before being used for methods or compositions described herein, the amplification of target nucleic acid is optional. Therefore, before being used for some embodiments of the methods and compositions described herein, the target nucleic acid may not be amplified. The target nucleic acid may optionally be derived from a synthetic library. Synthetic nucleic acid may have natural DNA or RNA composition or may be its analog.

[0086] Universal adapter

[0087] The target nucleic acid used in the methods or compositions described herein includes a universal adapter attached to each end. The method for attaching a universal adapter to each end of a target nucleic acid used in the methods described herein is known to those skilled in the art. Connection can be performed by using standard library preparation techniques (Chesney et al. U.S. Patent Publication No. 2018 / 0305753A1) connected, or by using tag fragmentation of a transposase complex (Gunderson et al., WO 2016 / 130704).

[0088] In one embodiment, double-stranded target nucleic acids from a sample, such as a fragmented sample, are processed by first connecting the same universal adapter molecule ("mismatched adapter", whose general features are defined below and further described in Gormley et al., US 7,741,463 and Bignell et al., US 8,053,192) to the 5' and 3' ends of a double-stranded target nucleic acid (which can be a known, partially known or unknown sample). In one embodiment, the universal adapter includes a universal capture binding sequence necessary for immobilizing the target nucleic acid on an array for subsequent sequencing. In another embodiment, the universal adapter present at each end of the target nucleic acid is further modified using a PCR step prior to immobilization and sequencing. For example, an initial primer extension reaction is performed using a universal primer binding site, in which an extension product complementary to the two chains of each individual target nucleic acid is formed and a universal capture binding sequence is added. The resulting primer extension products and optionally their amplified copies together provide a library of modified target nucleic acids that can be immobilized and then sequenced. The term "library" refers to a collection of target nucleic acids containing known common sequences at their 3' and 5' ends, and may also be referred to as 3' and 5' modified libraries. The 3' end and optionally the 5' end of the universal adaptor attached to the target nucleic acid may contain a homogenous population or a heterogeneous population of universal capture binding sequences described herein.

[0089] The universal adapters used in the methods of the present disclosure are referred to as "mismatch" adapters because, as explained in detail herein, the adapters contain regions of sequence mismatch, ie, they are not formed by annealing of perfectly complementary polynucleotide strands.

[0090] Mismatched adapters for use herein are formed by annealing two partially complementary polynucleotide chains to provide at least one double-stranded region (also referred to as a double-stranded nucleic acid region) and at least one unmatched single-stranded region, also referred to as a region of a single-stranded non-complementary nucleic acid strand, when the two chains are annealed.

[0091] The "double-stranded region" of a universal adapter is a short double-stranded region, typically comprising 5 or more consecutive base pairs, formed by annealing two partially complementary polynucleotide chains. The term refers to a double-stranded region of a nucleic acid where the two chains are annealed, and does not imply any particular structural conformation. As used herein, the term "double-stranded" when used to refer to a nucleic acid molecule means that substantially all of the nucleotides in the nucleic acid molecule are hydrogen-bonded to complementary nucleotides. A partially double-stranded nucleic acid may have at least 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95% of its nucleotides hydrogen-bonded to complementary nucleotides.

[0092] It is generally advantageous that the double-stranded region is as short as possible without sacrificing functionality. In this context, "functionality" refers to the ability of the double-stranded region to form a stable duplex under standard reaction conditions for an enzyme-catalyzed nucleic acid ligation reaction, which conditions are well known to the skilled reader (e.g., incubation at a temperature in the range of 4°C to 25°C in a ligation buffer suitable for the enzyme), so that the two strands forming the universal adapter remain partially annealed during the ligation of the universal adapter to the target molecule. It is not absolutely necessary that the double-stranded region is stable under conditions commonly used in the annealing step of a primer extension or PCR reaction.

[0093] The double-stranded region of a universal adapter is usually the same in all universal adapters used in the connection. Because the universal adapter is connected to the two ends of each target molecule, the modified target nucleic acid flank can have complementary sequences derived from the double-stranded region of the universal adapter. The longer the double-stranded region and the complementary sequence derived therefrom in the modified target nucleic acid construct, the greater the possibility that the modified target nucleic acid construct can fold back in these regions of internal self-complementarity under the annealing conditions used in primer extension and / or PCR and base pair with itself. Therefore, it is generally preferred that the length of the double-stranded region is 20 or less, 15 or less or 10 or less base pairs to reduce this effect. The stability of the double-stranded region can be increased by including non-natural nucleotides that exhibit stronger base pairing than standard Watson-Crick base pairs, and thus potentially shortening its length.

[0094] In one embodiment, the two strands of the universal adaptor are 100% complementary in the double-stranded region. It should be understood that one or more nucleotide mismatches can be tolerated within the double-stranded region, provided that the two strands are able to form a stable duplex under standard ligation conditions.

[0095] Universal adapters for use herein may generally include a double-stranded region that forms the "ligatable" end of the adapter, such as the end that is ligated to a double-stranded target nucleic acid in a ligation reaction. The ligatable end of the universal adapter may be a blunt end, or in other embodiments, there may be a short 5' or 3' overhang of one or more nucleotides to facilitate / promote ligation. The 5' terminal nucleotide of the ligatable end of the universal adapter is generally phosphorylated to achieve a phosphodiester connection to the 3' hydroxyl on the target polynucleotide.

[0096] The term "mismatch region" refers to a region of a universal adapter, a region of a single-stranded non-complementary nucleic acid chain, wherein the sequences of the two polynucleotide chains forming the universal adapter exhibit a degree of non-complementarity such that the two chains cannot completely anneal to each other under standard annealing conditions for primer extension or PCR reactions. Under standard reaction conditions for enzyme-catalyzed ligation reactions, the mismatch region may exhibit a degree of annealing as long as the two chains are converted to single-stranded form under the annealing conditions in the amplification reaction.

[0097] It should be understood that "mismatched regions" are provided by different parts of the same two polynucleotide chains that form double-stranded regions. Mismatches in adapter constructs can take the form of one chain being longer than the other, so that there is a single-stranded region on one of the chains, or a sequence is used, which is selected so that the two chains do not hybridize, thereby forming single-stranded regions on the two chains. Mismatches can also take the form of "bubbles", in which the two ends of the universal adapter construct can hybridize with each other and form a duplex, while the central region cannot. Under the condition that the other parts of the same two chains are annealed to form one or more double-stranded regions, the chain portions that form mismatched regions do not anneal. For the avoidance of doubt, it should be understood that in the context of the present disclosure, the single-stranded or single-base overhangs at the 3' ends of the polynucleotide duplex that subsequently undergoes connection with the target sequence do not constitute a "mismatched region".

[0098] The lower limit of the length of the mismatch region is generally determined by function, such as the need to provide a sequence suitable for the following items: i) primer binding for primer extension, PCR and / or sequencing (e.g., binding of a primer to a universal primer binding site), or ii) binding of a universal capture binding sequence to a capture sequence to immobilize the modified target nucleic acid on a surface. In theory, there is no upper limit to the length of the mismatch region, but it is generally advantageous to minimize the overall length of the universal adapter, for example, to facilitate separation of unbound universal adapters from the modified target nucleic acid construct after the ligation step. Therefore, it is generally preferred that the length of the mismatch region should be less than 50, or less than 40, or less than 30, or less than 25 consecutive nucleotides.

[0099] The single-stranded non-complementary nucleic acid region comprises at least one universal capture binding sequence at the 3' end (see Figure 1B, universal capture binding sequence 150). The 3' end of the universal adaptor comprises a first universal capture binding sequence that will hybridize to a first capture sequence present on the capture nucleic acid. Figure 2 As shown, the nucleic acid 200 of the first capture nucleic acid population comprises a first capture sequence 210. A chain of a modified target nucleic acid 230 comprising a first universal capture binding sequence 250 at the 3' end of the single strand is shown to hybridize with the first capture sequence 210. It is the interaction between the first universal capture binding sequence 250 and the first capture sequence 210 that is changed in order to reduce affinity and encode kinetic delay to the target nucleic acid inoculated in the hole. The standard ExAmp method uses a universal capture binding sequence and a capture sequence that is fully complementary over the entire length of the capture sequence. The ExAmp method described herein uses a universal capture binding sequence that contains one or more mismatches, has a reduced length, or a combination thereof. The result of the mismatch and / or reduced length is a reduced affinity between the two sequences compared to the affinity of the two fully complementary full-length sequences. The reduced affinity leads to a reduction in amplification efficiency, wherein the resulting amplification efficiency is typically a function of the number of differences between the universal capture binding sequence and the capture sequence.

[0100] Optionally, the 5' end of the universal adaptor comprises a second universal capture binding sequence attached to each end of the target nucleic acid, wherein the second universal capture binding sequence will hybridize to a second capture sequence present on the capture nucleic acid. Figure 1B As shown, universal capture binding sequence 180. Therefore, unless otherwise stated, the following discussion on how to adjust the universal capture binding sequence to reduce affinity applies to both 3' and 5' universal capture binding sequences.

[0101] The 3' end of the capture sequence serves as the starting point for DNA synthesis by the DNA polymerase in the methods described herein. Those skilled in the art will recognize that the nucleotides at the 3' end of the capture sequence and the corresponding nucleotides in the universal capture binding sequence should be complementary to maintain the ability of the DNA polymerase to initiate DNA synthesis.

[0102] The universal capture binding sequence may include one or more nucleotides that are not complementary to the capture sequence. In one embodiment, the universal capture binding sequence may include 1 to 5 mismatched nucleotides (also referred to as non-complementary nucleotides) compared to the capture sequence used in the amplification reaction described herein, such as at least 1, at least 2, at least 3, at least 4 or 5 mismatched nucleotides. The mismatched nucleotides may be wobble mismatches or true mismatches.

[0103] A wobble mismatch refers to a position in a population of universal capture binding sequences where all four nucleotides are represented. For example, if N is the wobble nucleotide in ACTNGC, then the population of universal capture binding sequences will include ACTTGC, ACTAGC, ACTCGC, and ACTGGC, and 25% of the universal capture binding sequences in the population will be complementary to the corresponding capture sequence nucleotides. In one embodiment, the universal capture binding sequence may include 1 to 5 wobble nucleotides, such as at least 1, at least 2, at least 3, at least 4, or 5 wobble nucleotides, compared to the capture sequence used in the amplification reaction described herein. In one embodiment, the wobble nucleotide may be located at any position throughout the universal capture binding sequence.

[0104] A true mismatch refers to a position that represents only three of the four nucleotides at a specific position in a population of universal capture binding sequences. For example, if G is the position of a true mismatch nucleotide in ACTTGC, the population of universal capture binding sequences will include ACTTCC, ACTTTC, and ACTTAC, and none of the universal capture binding sequences in the population are complementary to the corresponding nucleotide, in this example, C of the capture sequence. In one embodiment, the universal capture binding sequence may include 1 to 5 mismatched nucleotides, such as at least 1, at least 2, at least 3, at least 4, or 5 wobble nucleotides, compared to the capture sequence used in the amplification reaction described herein. In one embodiment, the wobble nucleotide may be located at any position throughout the universal capture binding sequence.

[0105] The skilled person will recognize that the use of wobble mismatches or true mismatches provides greater control over changing the affinity of the universal capture binding sequence. Using a universal capture binding sequence with only a single wobble nucleotide results in 25% of the universal capture binding sequences having complementarity at that position, having greater affinity than the other 75%, and expected higher amplification efficiency than the other 75%. Using a universal capture binding sequence with only a single true mismatch nucleotide results in all universal capture binding sequences having no complementarity at that position, reduced affinity, and expected reduced amplification efficiency.

[0106] In another embodiment, the universal capture binding sequence has a shortened length, which results in an affinity less than the affinity between the full-length universal capture binding sequence and the capture sequence. Capture sequences useful in standard amplification methods described herein typically have a length of about 20 to about 30 nucleotides, although if necessary, they can be longer or shorter. Universal capture binding sequences useful in methods described herein have a length that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides shorter than the capture sequence used in amplification reactions described herein. In one embodiment, the length of the universal capture binding sequence is reduced by removing nucleotides from the 3' end of the first universal capture binding sequence and / or from the 5' end of the second universal capture binding sequence.

[0107] The amplification reactions described herein can use a heterogeneous population of universal capture binding sequences (e.g., a plurality of different target nucleic acids can include a heterogeneous population of universal capture binding sequences present at the 3' end and optionally at the 5' end). In one embodiment, the heterogeneous population includes individual universal capture binding sequences with mismatched nucleotides. In one embodiment, the universal capture binding sequence has 1, 2, 3, 4, or 5 mismatched nucleotides. The mismatched nucleotides can be wobble mismatches, true mismatches, or a combination thereof.

[0108] In one embodiment, the heterogeneous population comprises individual universal capture binding sequences with shortened lengths. In one embodiment, the heterogeneous population comprises individual universal capture binding sequences that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides shorter than the capture sequences used in the amplification reactions described herein.

[0109] In one embodiment, the heterogeneous population comprises individual universal capture binding sequences having a combination of one or more mismatched nucleotides and shortened length. The number of mismatched nucleotides and the number of nucleotides missing from the universal capture binding sequence can be present in any combination, for example, the number of mismatched nucleotides and the number of missing nucleotides are independent.

[0110] The heterogeneous population may also include individual target nucleic acids having a universal capture binding sequence with 100% complementarity to the capture sequence at its 3' end and optionally at its 5' end. The molar ratios of different universal capture binding sequences in the heterogeneous population may be equal or varied. In those embodiments where the molar ratios are not equal, a higher molar ratio of those universal capture binding sequences with higher amplification efficiency is preferred. Therefore, in those embodiments where the heterogeneous population includes a universal capture binding sequence with 100% complementarity to the capture sequence, the universal capture binding sequence with 100% complementarity may be present in a greater proportion than any other member of the heterogeneous population.

[0111] The single-stranded non-complementary nucleic acid region also typically comprises at least one universal primer binding site. A universal primer binding site is a universal sequence that can be used to amplify and / or sequence a target nucleic acid that is attached to a universal adaptor.

[0112] The single-stranded non-complementary nucleic acid chain region may also include at least one index. The index may be used as a characteristic marker of a specific target nucleic acid source on the array. Typically, the index is a synthetic nucleotide sequence that is a part of a universal adapter that is added to the target nucleic acid as part of a library preparation step. Therefore, the index is a nucleic acid sequence attached to each target molecule of a specific sample, and its presence indicates or is used to identify a sample or source from which a target molecule was isolated.

[0113] Preferably, the index length is up to 20 nucleotides, more preferably 1-10 nucleotides, and most preferably 4-8 nucleotides in length. For example, a tetranucleotide index provides multiplexing of 256 (4 4 ) samples, while the six-base index can handle 4,096 (4 6 ) samples.

[0114] In one embodiment, when the universal capture binding sequence is ligated to the double-stranded target fragment, it is part of the universal adapter, and in another embodiment, after the universal adapter is ligated to the double-stranded target fragment, the universal primer extension binding site is added to the universal adapter. Conventional methods can be used to complete the addition, including PCR-based methods.

[0115] The precise nucleotide sequence of the universal adaptor is generally not important to the invention and can be selected by the user so that the desired sequence elements are ultimately included in the common sequence of multiple different modified target nucleic acids, for example, to provide a universal capture binding sequence and binding sites for a particular set of universal amplification primers and / or sequencing primers. Other sequence elements can be included, for example, to provide binding sites for sequencing primers, for example on a solid support, which will ultimately be used to sequence the target nucleic acids in the library or products derived from amplification of the target nucleic acids in the library.

[0116] Although the precise nucleotide sequence of the universal adaptor is generally not limited to the present disclosure, the sequence of the individual strands in the mismatched region should be such that none of the individual strands exhibit any internal self-complementarity, which under standard annealing conditions could result in self-annealing, formation of hairpin structures, etc. Self-annealing of strands in the mismatched region should be avoided because it can prevent or reduce specific binding of an amplification primer to this strand.

[0117] Mismatched adaptors are preferably formed from two DNA strands, but may include a mixture of natural and non-natural nucleotides (eg, one or more ribonucleotides) linked by a mixture of phosphodiester and non-phosphodiester backbone linkages.

[0118] Connect and amplify

[0119] The method of connection is known in the art and uses standard methods. Such methods use ligase, such as DNA ligase to realize or catalyze the connection of the ends of the two polynucleotide chains of the universal adapter and double-stranded target nucleic acid in this case, thereby forming a covalent connection. The universal adapter may contain a 5'-phosphate moiety to promote the connection with the 3'-OH present on the target fragment. The double-stranded target nucleic acid contains a 5'-phosphate moiety, which is residual from the shearing process or is added using an enzyme treatment step, and has been end-repaired, and optionally extended by one or more protruding bases, thereby providing a 3'-OH suitable for connection. In this context, connection refers to the covalent connection of a polynucleotide chain that was not previously covalently connected. In particular aspects of the present disclosure, such connection occurs by forming a phosphodiester connection between two polynucleotide chains, but other covalently connected modes (such as non-phosphodiester main chain connections) can be used.

[0120] As discussed herein, in one embodiment, the universal adaptor used for ligation is complete and includes a universal capture binding sequence and other universal sequences, such as a universal primer binding site and an index sequence.The resulting plurality of target nucleic acids can be used to prepare an immobilized sample for sequencing.

[0121] Also as discussed herein, in one embodiment, the universal adapter for connection comprises a universal primer binding site and an index sequence, and does not comprise a universal capture binding sequence. The resulting multiple modified target nucleic acids can be further modified to include specific sequences, such as universal capture binding sequences. Methods for adding specific sequences, such as universal capture binding sequences, to universal primers connected to double-stranded target fragments include PCR-based methods, and are known in the art, and are described, for example, in: Bignell et al. (US 8,053,192) and Gunderson et al. (WO2016 / 130704).

[0122] In those embodiments where the universal linker is modified, an amplification reaction is prepared. The contents of the amplification reaction are known to those skilled in the art, and include suitable substrates (e.g., dNTPs), enzymes (e.g., DNA polymerases), and buffer components required for the amplification reaction. Typically, the amplification reaction requires at least two amplification primers, typically expressed as "forward" and "reverse" primers (primer oligonucleotides), which can specifically anneal with a portion of a polynucleotide sequence to be amplified (e.g., a target nucleic acid) under the conditions encountered in the primer annealing step of each cycle of the amplification reaction. It should be understood that if the primer contains any nucleotide sequence that does not anneal with the modified target nucleic acid in the first amplification cycle, this sequence can be copied into the amplification product. For example, using a primer with a universal capture binding sequence, such as a primer that does not anneal with a modified target nucleic acid, the universal capture binding sequence will be incorporated into the resulting amplicon.

[0123] Amplification primers are typically single-stranded polynucleotide structures. They may also contain mixtures of natural and non-natural bases and natural and non-natural backbone connections, provided that any non-natural modification does not exclude the function of a primer, defined as the ability to anneal to a template polynucleotide chain during the conditions of the amplification reaction and to act as the starting point for synthesizing a new polynucleotide chain complementary to the template chain. In addition, primers may include non-nucleotide chemical modifications, such as phosphorothioates to increase exonuclease resistance, provided again that the modification does not prevent primer function.

[0124] Preparation of immobilized samples for sequencing

[0125] The method disclosed herein may include reacting amplification reagents (arrays of amplification sites and multiple different modified target nucleic acids) to produce multiple amplification sites, each of which includes a clonal population of amplicons from individual target nucleic acids that have been inoculated at the site. In a standard reaction, amplification occurs due to a relatively slow rate (e.g., relatively slow diffusion or transport) of target nucleic acid inoculation relative to a relatively fast rate of amplification to fill the site with nucleic acid seed copies. In the method described herein, amplification is excluded due to a kinetic delay in the formation of the first copy of the target nucleic acid inoculated at the site relative to a relatively fast rate of producing subsequent copies to fill the site. For example, individual sites may have been inoculated with several different target nucleic acids, and each target nucleic acid has different universal capture binding sequences (such as a plurality of different modified target nucleic acids comprising a heterogeneous population of universal capture binding sequences). However, it is expected that the first copy of any given target nucleic acid is formed depending on the amplification efficiency of its universal capture binding sequence, so that the average rate of the first copy formation is relatively slow compared to the rate of producing subsequent copies. In this case, although individual sites may have been inoculated with several different target nucleic acids, only one will start amplification first, and exclusion amplification generally only allows the target nucleic acid to fill the amplification site. More specifically, once a first target nucleic acid begins to amplify, the site will quickly fill to capacity with its copies, thereby preventing the formation of copies of the second target nucleic acid at the site.

[0126] In some embodiments, even before the second target nucleic acid begins to amplify at this site, the amplification site is not filled to capacity, and obvious clonality can be achieved. Under certain conditions, the amplification of the first target nucleic acid can proceed to the following point, i.e., produce a sufficient number of copies to effectively outperform or overwhelm the copy generation of the second target nucleic acid transported to this site. For example, in the embodiment of using the bridge amplification process on the circular feature of diameter less than 500nm, it has been determined that after the circulation of 14 rounds of exponential amplification is carried out to the first target nucleic acid, the pollution from the second target nucleic acid at the same site will produce insufficient contaminant amplicon to adversely affect the synthesis sequencing analysis on the Illumina sequencing platform.

[0127] In all embodiments, the amplification sites in the array do not need to be completely cloned. On the contrary, for some applications, the independent amplification sites can be mainly filled with the amplicon from the first target nucleic acid, and can also have low-level contamination amplicon from the second target nucleic acid. The array can have one or more amplification sites with low-level contamination amplicon, as long as the contamination level does not produce unacceptable influence on the subsequent use of the array. For example, when the array is to be used for detection application, the acceptable contamination level is the level that does not affect the resolution of the signal-to-noise ratio or detection technology in an unacceptable manner. Therefore, the apparent clonality is usually related to the specific use or application of the array made by the methods described herein. For specific applications, the exemplary level of contamination acceptable at the independent amplification site includes but is not limited to 0.1%, 0.5%, 1%, 5%, 10% or 25% of the contamination amplicon at most. The array can include one or more amplification sites with these exemplary levels of contamination amplicon. For example, up to 5%, 10%, 25%, 50%, 75% or even 100% of the amplification sites in the array can have some contamination amplicon.

[0128] Although the above embodiment that is present in the amplification site for target nucleic acid before amplification illustrates the first amplicon and subsequent amplicon formation that utilize differentially active primers to cause different speeds, the method can also be carried out under the condition that target nucleic acid is transported (for example, by diffusion) to the amplification site when amplification occurs.Therefore, the elimination amplification method can utilize both the relatively slow transport rate and the relatively slow generation of the first amplicon with respect to the subsequent amplicon formation.Therefore, amplification reaction as herein described can be carried out so that target nucleic acid is transferred to the amplification site from solution simultaneously with the following items: (i) produce the first amplicon, and (ii) produce subsequent amplicon at other sites of the array.In a specific embodiment, the average rate of the subsequent amplicon produced at the amplification site can exceed the average rate at which the target nucleic acid is transported to the amplification site from solution.In some cases, sufficient number of amplicon can be produced from the single target nucleic acid at the independent amplification site to fill the capacity of the corresponding amplification site.The rate at which the amplicon is produced to fill the capacity of the corresponding amplification site can for example exceed the rate at which the independent target nucleic acid is transported to the amplification site from solution.

[0129] The amplification reagent used in the method described herein is preferably capable of quickly producing a copy of the target nucleic acid at the amplification site. Typically, the amplification reagent used in the disclosed method will include a polymerase and nucleoside triphosphates (NTPs). Any of a variety of polymerases known in the art can be used, but in some embodiments, a polymerase with a negative exonuclease may be preferably used. For embodiments that produce DNA copies, NTPs may be deoxyribonucleotide triphosphates (dNTPs). Typically, four natural species dATP, dTTP, dGTP, and dCTP will be present in DNA amplification reagents; however, analogs may be used if desired. For embodiments that produce RNA copies, NTPs may be ribonucleotide triphosphates (rNTPs). Typically, four natural species rATP, rUTP, rGTP, and rCTP will be present in RNA amplification reagents; however, analogs may be used if desired.

[0130] Amplification reagents may include other components that contribute to amplicon formation and in some cases improve the rate of amplicon formation. An example is a recombinase load protein. Recombinases can promote amplicon formation by allowing repeated invasion / extension. More specifically, recombinases can use target nucleic acids as templates for amplicon formation to promote polymerase invasion of target nucleic acids and polymerase extension of primers. This process can be repeated as a chain reaction, wherein the amplicon produced by each round of invasion / extension is used as a template in subsequent rounds. Since no denaturation cycle (such as by heating or chemical denaturation) is required, this process will occur faster than standard PCR. Therefore, the amplification promoted by recombinases can be carried out isothermally. It is generally desirable to include ATP or other nucleotides (or in some cases its non-hydrolyzable analogs) in the amplification reagent promoted by recombinases to promote amplification. A mixture of recombinases, single-stranded binding (SSB) proteins and auxiliary proteins is particularly useful. Exemplary formulations for amplification promoted by recombinases include those sold commercially by TwistDx (Cambridge, UK) with TwistAmp kits. Useful components and reaction conditions for recombinase-facilitated amplification reagents are described in US Pat. No. 5,223,414 and US Pat. No. 7,399,590, each of which is incorporated herein by reference.

[0131] Another example of a component that can be included in an amplification reagent to promote amplicon formation and in some cases improve the rate of amplicon formation is a helicase. A helicase can promote amplicon formation by a chain reaction that allows amplicon formation. Since no denaturation cycle (such as by heating or chemical denaturation) is required, the process can occur faster than standard PCR. Therefore, the amplification promoted by the helicase can be carried out isothermally. A mixture of a helicase and a single-stranded binding (SSB) protein is particularly useful because SSB can further promote amplification. Exemplary formulations for promoting amplification by a helicase include those sold commercially by Biohelix (Beverly, MA) with an IsoAmp test kit. In addition, examples of useful formulations including helicase proteins are described in U.S. Patent Nos. 7,399,590 and 7,829,284, each of which is incorporated herein by reference.

[0132] Another example of a component that can be included in an amplification reagent to facilitate amplicon formation, and in some cases increase the rate of amplicon formation, is an initiation binding protein.

[0133] The presence of a molecular crowding agent in the solution can be used to assist in exclusion amplification. Examples of useful molecular crowding agents include, but are not limited to, polyethylene glycol (PEG), dextran, or polyvinyl alcohol. Exemplary molecular crowding agents and formulations are described in U.S. Pat. No. 7,399,590.

[0134] The rate at which the amplification reaction occurs can be increased by increasing the concentration or amount of one or more active components of the amplification reaction. For example, the amount or concentration of a polymerase, nucleotide triphosphates, primers, recombinases, helicases or SSB can be increased to increase the amplification rate. In some cases, one or more active components of the amplification reaction that increase in amount or concentration (or otherwise operate in the methods described herein) are non-nucleic acid components of the amplification reaction.

[0135] The amplification rate can also be increased in the methods described herein by regulating the temperature. For example, the amplification rate at one or more amplification sites can be increased by raising the temperature at one or more sites to the maximum temperature at which the reaction rate is reduced due to denaturation or other adverse events. The optimum temperature or desired temperature can be determined empirically from the known properties of the amplification components used or for a given amplification reaction mixture. Such adjustments can be made empirically based on a priori predictions of the primer melting temperature (Tm) or empirically.

[0136] The rate at which an amplification reaction occurs can be increased by increasing the activity of one or more amplification reagents. For example, a cofactor that increases the extension rate of a polymerase can be added to a reaction using that polymerase. In some embodiments, a metal cofactor such as magnesium, zinc, or manganese can be added to the polymerase reaction or betaine can be added.

[0137] In some embodiments of the method set forth herein, it is desirable to use a double-stranded target nucleic acid population. It has been observed that, under conditions of excluding amplification, it is effective for double-stranded target nucleic acids to form amplicons in the site array. For example, in the presence of recombinase and single-stranded binding proteins, multiple amplification sites (compared to single-stranded target nucleic acids of the same concentration) of a clonal population with amplicons can be more effectively produced by double-stranded target nucleic acids. However, it will be understood that single-stranded target nucleic acids can be used in some embodiments of the method set forth herein.

[0138] The method described herein can use any of a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA) or random priming amplification (RPA). In some embodiments, for example, when the amplification site can contain an amplicon with a volume of the desired capacity, amplification can be performed in a solution. Preferably, the amplification techniques used under the exclusion of amplification conditions in the disclosed method will be performed on a solid phase. For example, one or more primers for amplification can be attached to a solid phase at the amplification site. In a PCR embodiment, one or both of the primers for amplification can be attached to a solid phase. The form of using two primers attached to the surface is generally referred to as bridge amplification, because the double-stranded amplicon forms a bridge-like structure between the primers attached to the two surfaces, and the primer is flanked by the template sequence that has been replicated. Exemplary reagents and conditions that can be used for bridge amplification are described in, e.g., U.S. Patent No. 5,641,658; U.S. Patent Publication No. 2002 / 0055100; U.S. Patent No. 7,115,400; U.S. Patent Publication No. 2004 / 0096853; U.S. Patent No. 2004 / 0002090; U.S. Patent Publication No. 2007 / 0128624; and U.S. Patent Publication No. 2008 / 0009420. Solid phase PCR amplification can also be performed using one of the amplification primers attached to a solid support and a second primer in solution. An exemplary form of a combination of surface-attached primers and soluble primers is emulsion PCR, described in, for example, Dressman et al., Proc. Natl. Acad. Sci. USA 100: 8817-8822 (2003), WO 05 / 010145, or U.S. Patent Publication No. 2005 / 0130173 or 2005 / 0064460. Emulsion PCR is an example of this form, and it should be understood that for the purposes of the methods described herein, the use of emulsion is optional, and in fact, for some embodiments, emulsion is not used. The described PCR technique can be modified for non-cyclic amplification (such as isothermal amplification), which uses components exemplified elsewhere herein to promote or increase amplification rate. Therefore, the described PCR technique can be used under exclusion amplification conditions.

[0139] The RCA technique can be improved for the method of the present disclosure. The exemplary components that can be used in the RCA reaction and the principle of RCA producing amplicons are described in, for example, Lizardi et al., Nat.Genet.19:225-232 (1998) and US2007 / 0099208 A1. The primers used for RCA can be attached to the solid support surface in solution or at the amplification site. The RCA technique exemplified in the above references can be improved according to the teachings of this article, for example, to improve the amplification rate to be suitable for specific applications. Therefore, the RCA technique can be used under the exclusion of amplification conditions.

[0140] The MDA technique can be modified for use in the methods disclosed herein. Some basic principles and useful conditions of MDA have been described, for example, in Dean et al., Proc Natl. Acad. Sci. USA 99: 5261-66 (2002); Lage et al., Genome Research 13: 294-307 (2003); Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20: 1691-96 (1992); U.S. Patent No. 5,455,166; U.S. Patent No. 5,130,238; and U.S. Patent No. 6,214,587. Primers for MDA can be attached to a solid support surface in solution or at the amplification site. The MDA technique exemplified in the above references can be modified according to the teachings of this article, for example, to increase the amplification rate to suit a specific application. Therefore, the MDA technique can be used under exclusion amplification conditions.

[0141] In a specific embodiment, the combination of described amplification techniques can be used to prepare arrays under exclusion amplification conditions. For example, RCA and MDA can be used in combination, wherein RCA is used to produce serial amplicons in solution (for example, using solution phase primers). It can then be used as a template to carry out MDA using primers that are attached to a solid support surface at the amplification site. In this example, the amplicon produced after the combination RCA and MDA steps will be attached to the surface of the amplification site.

[0142] As exemplified with respect to the above several embodiments, the method disclosed herein does not need to use cyclic amplification techniques. For example, the amplification of the target nucleic acid can be performed at an amplification site without a denaturation cycle. An exemplary denaturation cycle includes introducing a chemical denaturant into the amplification reaction and / or increasing the temperature of the amplification reaction. Therefore, the amplification of the target nucleic acid does not need to include the step of replacing the amplification solution with a chemical reagent that denatures the target nucleic acid and the amplicon. Similarly, the amplification of the target nucleic acid does not need to include heating the solution to a temperature that denatures the target nucleic acid and the amplicon. Therefore, amplification of the target nucleic acid at the amplification site can be performed isothermally within the duration of the method described herein. In fact, the amplification method described herein can occur without performing one or more cyclic operations on certain amplification techniques under standard conditions. In addition, in some standard solid phase amplification techniques, washing is performed after the target nucleic acid is loaded onto the substrate and before the amplification begins. However, in an embodiment of the present method, a washing step is not required between transporting the target nucleic acid to the reaction site and amplifying the target nucleic acid at the amplification site. On the contrary, transport (such as by diffusion) and amplification are allowed to occur simultaneously to provide exclusion amplification.

[0143] In some embodiments, it may be desirable to repeat the amplification cycles that occur under the exclusion amplification conditions. Therefore, although a copy of the target nucleic acid can be prepared at a single amplification site without the need for cyclic operation, the array of the amplification site can be cyclically processed to increase the number of sites containing amplicons after each cycle. In a specific embodiment, the amplification conditions can be changed from one cycle to the next cycle. For example, the above-mentioned one or more conditions that change the transport rate or change the amplification rate can be adjusted between cycles. Like this, the transport rate can be increased from one cycle to another cycle, the transport rate can be reduced from one cycle to another cycle, the amplification rate can be increased from one cycle to another cycle, or the amplification rate can be reduced from one cycle to another cycle.

[0144] Composition

[0145] During or after the amplification clustering method described herein, different compositions can be produced. In one embodiment, the composition includes an array of amplification sites. Each site comprises a first and a second capture nucleic acid, which comprises a first and a second capture sequence, respectively, wherein the first and second capture nucleic acids are bound to the surface of the site. Different sites of the array comprise target nucleic acids hybridized with the first capture sequence of the first capture nucleic acid. The target nucleic acids at different sites each comprise a universal capture binding sequence hybridized with the capture sequence at the 3' end. The universal capture binding sequence present has a lower affinity for the capture sequence than the universal capture binding sequence with 100% complementarity to the first capture sequence. In one embodiment, different universal capture binding sequences are present at each site, for example, there is a first heterogeneous population of universal capture binding sequences. The first heterogeneous population may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 different universal capture binding sequences.

[0146] In one embodiment, the first universal capture binding sequence comprises 1 to 5 nucleotides that are not complementary to the first capture sequence. The composition may include some target nucleic acids having a universal capture binding sequence that is 100% complementary to the first capture sequence. In one embodiment, members of the first heterogeneous population that are 100% complementary to the first capture sequence are present in a greater number than other members of the first heterogeneous population. The first heterogeneous population may also include individual first universal capture binding sequences that are less than the length of the first capture sequence, for example, 1 to 12 nucleotides less in length than the length of the first capture sequence. The length of individual members of the first heterogeneous population may have a length less than the length of the first capture sequence and comprise 1 to 5 nucleotides that are not complementary to the sequence of the first capture sequence, or may be 100% complementary to the sequence of the first capture sequence.

[0147] Optionally, the 5' end may comprise a second universal capture binding sequence having a complement having a lower affinity for the second capture sequence than the second universal capture binding sequence having a complement that is 100% complementary to the second capture sequence. In one embodiment, the complement of the second universal capture binding sequence comprises 1 to 5 nucleotides that are not complementary to the second capture sequence. The composition may comprise some target nucleic acids having a second universal capture binding sequence having a complement that is 100% complementary to the second capture sequence. In one embodiment, different second universal capture binding sequences are present at each site, for example, a second heterogeneous population of second universal capture binding sequences is present. The second heterogeneous population may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 different second universal capture binding sequences. In one embodiment, members of the second heterogeneous population having a complement that is 100% complementary to the second capture sequence are present in greater numbers than other members of the second heterogeneous population. The second heterogeneous population of universal capture binding sequences at the 5' end may also include individual second universal capture binding sequences having a length less than the length of the second capture sequence, for example, 1 to 12 nucleotides less than the length of the second capture sequence. Individual members of the second heterogeneous population may have a length less than the length of the second capture sequence and include complements having 1 to 5 nucleotides that are not complementary to the sequence of the second capture sequence, or 100% complementary to the sequence of the second capture sequence.

[0148] Another composition that can be produced includes a solution comprising different double-stranded target nucleic acids from a single sample or source (e.g., a library), wherein each target nucleic acid comprises a universal adapter attached at each end. The universal adapter comprises a universal capture binding sequence, and the universal capture binding sequence is a heterogeneous population. The heterogeneous population may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 different universal capture binding sequences.

[0149] Application in Sequencing / Sequencing Methods

[0150] For example, arrays of the present disclosure that have been produced by methods described herein and include amplified target nucleic acids at amplification sites can be used for any of a variety of applications. Particularly useful applications are nucleic acid sequencing. An example is sequencing-by-synthesis (SBS). In SBS, monitoring nucleic acid primers extend along a nucleic acid template (e.g., a target nucleic acid or its amplicon) to determine the sequence of nucleotides in the template. Potential chemical processes can be polymerization reactions (e.g., as catalyzed by polymerase). In specific SBS embodiments based on polymerase, fluorescently labeled nucleotides are added to primers (thus extending primers) in a template-dependent manner, so that the sequence of the template can be determined using the order and type of nucleotides added to the primers. A plurality of different templates at different sites of arrays described herein can be subjected to SBS techniques under conditions where events occurring for different templates can be distinguished due to their position in the array.

[0151] The flow cell provides a convenient form for accommodating an array, which is produced by the method disclosed herein, and is subjected to SBS or other detection techniques, which involve repeated delivery of reagents in a cycle. For example, in order to start the first SBS cycle, one or more labeled nucleotides, DNA polymerases, etc. can be flowed into / flowed into the flow cell accommodating the nucleic acid template array. Those sites of the array where primer extension causes incorporation of labeled nucleotides can be detected. Optionally, the labeled nucleotides can further include a reversible termination property, and once nucleotides have been added to the primer, the reversible termination property terminates further primer extension. For example, a nucleotide analog with a reversible terminator portion can be added to the primer so that subsequent extension does not occur until a deblocking agent is delivered to remove the portion. Therefore, for an embodiment using reversible termination, the deblocking agent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between each delivery step. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with arrays produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; U.S. Patent No. 7,057,026; WO 91 / 06678; WO07 / 123,744; U.S. Patent No. 7,329,492; U.S. Patent No. 7,211,414; U.S. Patent No. 7,315,019; U.S. Patent No. 7,405,281, and U.S. Patent No. 8,343,746.

[0152] Other sequencing programs using cyclic reactions can be used, such as pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi, et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al. Science 281 (5375), 363 (1998); U.S. Patent No. 6,210,891; 6,258,568 and 6,274,320). In pyrophosphate sequencing, the released PPi can be detected by being immediately converted into adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP produced can be detected by photons generated by luciferase. Therefore, the sequencing reaction can be monitored by a luminescent detection system. The pyrophosphate sequencing program does not necessarily require an excitation radiation source for a fluorescence-based detection system. Useful fluidic systems, detectors, and procedures for applying pyrosequencing to the arrays of the present disclosure are described in, for example, WIPO Published Patent Applications 2012 / 058096, US 2005 / 0191698 Al, US Patent No. 7,595,883, and US Patent No. 7,244,559.

[0153] Ligation sequencing reactions are also useful, including, for example, those described in Shendure et al. Science 309:1728-1732 (2005); U.S. Pat. No. 5,599,675; and U.S. Pat. No. 5,750,341. Some embodiments may include hybridization sequencing procedures, such as those described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO 1989 / 10977. In both ligation sequencing and hybridization sequencing procedures, repeated cycles of oligonucleotide delivery and detection are performed on template nucleic acids (e.g., target nucleic acids or amplicons thereof) present at array sites. Fluid systems for SBS methods as described herein or described in the references cited herein can be easily adapted to deliver reagents for ligation sequencing or hybridization sequencing procedures. Typically, oligonucleotides are fluorescently labeled and can be detected using a fluorescence detector that is similar to the fluorescence detector described for the SBS procedures in this article or the references cited herein.

[0154] Some embodiments may use methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide or using a zero-mode waveguide. Techniques and reagents for FRET-based sequencing are described in, for example, Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008).

[0155] Some SBS embodiments include detecting the protons released after the nucleotides are incorporated into the extension products. For example, sequencing based on the detection of the released protons can use electrical detectors and related technologies available from Ion Torrent (Guilford, Conn., a Life Technologies subsidiary) or US 2009 / 0026082 A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143 A1; or sequencing methods and systems described in US 2010 / 0282617 A1. The methods described herein for amplifying target nucleic acids using exclusion amplification can be easily applied to substrates for detecting photons. More specifically, the methods described herein can be used for clonal populations of amplicons at sites of arrays for detecting photons.

[0156] For example, a useful application of the array of the present disclosure that has been produced by the methods described herein is gene expression analysis. RNA sequencing techniques, such as those referred to as digital RNA sequencing, can be used to detect or quantify gene expression. RNA sequencing techniques can be performed using sequencing methods known in the art, such as the above-mentioned methods. It is also possible to detect or quantify gene expression using hybridization techniques performed by direct hybridization with the array or using multiple assays that detect its products on the array. For example, the array of the present disclosure that has been produced by the methods described herein can also be used to determine the genotype of a genomic DNA sample from one or more individuals. Exemplary methods for array-based expression and genotyping analysis that can be performed on the array of the present disclosure are described in U.S. Patent Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or U.S. Patent Publication Nos. 2005 / 0053980 A1; 2009 / 0186349 A1 or US 2005 / 0181440 A1.

[0157] Another useful application of arrays produced by the methods described herein is single cell sequencing. When combined with indexing methods, single cell sequencing can be used in chromatin accessibility assays to generate profiles of active regulatory elements in thousands of single cells, and single cell whole genome libraries can be generated. Examples of single cell sequencing that can be performed on arrays disclosed herein are recorded in U.S. published patent application 2018 / 0023119 A1, U.S. provisional application serial numbers 62 / 673,023 and 62 / 680,259.

[0158] The advantage of the methods described herein is that they provide any one of multiple nucleic acid libraries to quickly and effectively create arrays. Therefore, the present disclosure provides an integrated system, which can use one or more methods set forth herein to prepare arrays and can also use technology known in the art (such as those illustrated above) to detect nucleic acids on arrays. Therefore, the integrated system of the present disclosure can include a fluid component that can deliver amplification reagents to the amplification site array, such as a pump, a valve, a reservoir, a fluid line, etc. Particularly useful fluid components are flow cells. The flow cell can be configured and / or used in the integrated system to create an array of the present disclosure and detect the array. Exemplary flow cells are described in, for example, US2010 / 0111768A1 and U.S. Patent No. 8,951,781. As illustrated by the flow cell, one or more fluid components of the integrated system can be used for amplification method and detection method. Taking nucleic acid sequencing embodiment as an example, one or more fluid components of the integrated system can be used for amplification method described herein and for the delivery of sequencing reagents in sequencing methods such as the sequencing methods illustrated above. Alternatively, the integrated system can include a separate fluid system to carry out amplification method and carry out detection method. Examples of integrated sequencing systems capable of creating nucleic acid arrays and determining nucleic acid sequences include, but are not limited to, MiSeq TM 、HiSeq TM 、NextSeq TM 、MiniSeq TM 、NovaSeq TM and iSeq TM The apparatus described in the ELISA platform (Illumina, Inc., San Diego, Calif.) and in U.S. Patent No. 8,951,781 can be modified according to the teachings set forth herein to prepare arrays that use exclusion amplification.

[0159] The system capable of performing the methods described herein need not be integrated with the detection device. Instead, it can be an independent system or a system integrated with other devices. In such embodiments, fluidic components similar to those exemplified above in the context of an integrated system can be used.

[0160] Whether integrated with detection capability or not, a system capable of performing the method proposed herein may include a system controller capable of executing a set of instructions to perform one or more steps of the method, technique or process described herein. For example, the instruction may guide the step of creating an array under bridge amplification conditions. Optionally, the instruction may further guide the step of detecting nucleic acids using the method previously described herein. Useful system controllers may include any processor-based or microprocessor-based system, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), logic circuits, and any other circuits or processors capable of performing the functions described herein. A set of instructions for a system controller may be in the form of a software program. As used herein, the terms "software" and "firmware" are interchangeable and include any computer program stored in a memory for execution by a computer, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory. Software may take various forms, such as system software or application software. In addition, software may take the form of a collection of separate programs, or a program module or a portion of a program module within a larger program. Software may also include modular programming in the form of object-oriented programming.

[0161] Several applications of the disclosed array are illustrated above in the context of overall detection, wherein multiple amplicons present at each amplification site are detected together. In selectable embodiments, single nucleic acids can be detected at each amplification site, whether it is a target nucleic acid or an amplicon thereof. For example, the amplification site can be configured to comprise a single nucleic acid molecule and multiple filling nucleic acids with a target nucleotide sequence to be detected. In this example, the function of filling nucleic acid is to fill the capacity of the amplification site, and they are not necessarily intended to be detected. Single molecules to be detected can be detected by a method that can distinguish single molecules in the background of filling nucleic acid. Any of various single molecule detection techniques can be used, including, for example, improvements to the above-mentioned set detection techniques, to detect incremented sites, or using more sensitive markers to detect sites. Other examples of single molecule detection methods that can be used are shown in US 2011 / 0312529A1; U.S. Patent No. 9,279,154; and US 2013 / 0085073 A1.

[0162] One or more methods described herein can be used to create an array that can be used for single molecule nucleic acid detection when there is the following modification.A plurality of different target nucleic acids can be configured to include both target nucleotide sequences to be detected and one or more filling nucleotide sequences to be amplified to produce filling amplicons.A plurality of different target nucleic acids can be included in amplification reagents such as those described elsewhere herein, and react with the amplification site array under the exclusion of amplification conditions so that the filling nucleotide sequence fills the amplification site.Can be used to allow the filling sequence to amplify, and the exemplary configuration that forbids the target sequence amplification includes, for example, a single target molecule with a first region and a second region, wherein the first region has a filling sequence with a binding site for the amplification primer present in the amplification site on the flank, and the second region has a target sequence outside the flanking region.In another configuration, the target nucleic acid can include a single molecule or chain with a target sequence and a filling sequence respectively.The separated molecule or chain can be attached to a particle or formed with the arm of a nucleic acid dendrimer or other branched structures.

[0163] In a specific embodiment, an array having an amplification site that includes a filler sequence and a target sequence can be detected using a primer extension assay or synthetic sequencing technology. In this case, specific extension can be achieved on a target nucleotide sequence rather than a large number of filler sequences by using a suitably placed primer binding site. For example, the binding site for a sequencing primer can be placed upstream of the target sequence and may not be in any filler sequence. Alternatively or in addition, the target sequence may include one or more non-natural nucleotide analogs that cannot be hydrogen-bonded with standard nucleotides. Non-natural nucleotides can be placed downstream of the primer binding site (for example, in the target sequence or between the target sequence and the region of the primer binding site), so extension or synthetic sequencing will be prevented before adding a suitable nucleotide chaperone (i.e., a nucleotide chaperone that can hydrogen-bond with the non-natural analog in the target sequence). Nucleotide analogs isocytosine (isoC) and isoguanine (isoG) are particularly useful because they are specifically paired with each other, but not with other standard nucleotides used in most extension and synthetic sequencing technologies. Another benefit of using isoC and / or isoG at or upstream of the target sequence is that it prevents unwanted amplification of the target sequence during the amplification step by omitting the corresponding chaperone from the nucleotide mix used for amplification.

[0164] It should be understood that, for example, arrays of the present disclosure that have been produced by the methods described herein need not be used for detection methods. On the contrary, the array can be used to store nucleic acid libraries. Therefore, the array can be stored in a state where nucleic acids are preserved. For example, the array can be stored in a dry state, a frozen state (e.g., in liquid nitrogen) or in a solution that protects nucleic acids. Alternatively or in addition, the array can be used to replicate a nucleic acid library. For example, the array can be used to create a repeat amplicon from one or more sites on the array.

[0165] Several embodiments of the present disclosure have been exemplified herein for transporting target nucleic acids to the amplification sites of arrays and producing copies of captured target nucleic acids at the amplification sites. Similar methods can be used for non-nucleic acid target molecules. Therefore, the methods set forth herein can be used together with other target molecules that replace exemplary target nucleic acids. For example, the methods of the present disclosure can be implemented to transport individual target molecules from different target molecule populations. Each target molecule can be transported to (and captured in some cases in) individual sites of the array to trigger a reaction at the capture site. The reaction at each site can, for example, produce a copy of the capture molecule, or the reaction can change the site to separate or isolate the captured molecule. In either case, the end result can be a site of an array, each of which is pure for the type of target molecule present in a population containing different types of target molecules.

[0166] In the specific embodiment using the target molecule other than nucleic acid, the library of different target molecules can be prepared using the method of exclusion amplification. For example, under the condition of randomly inoculating the site of the array with the target molecule in the solution and generating a copy of the target molecule to fill each inoculation site to capacity, the target molecule array can be prepared. According to the exclusion amplification method disclosed in the present invention, under the condition that the rate of generating copies exceeds the inoculation rate, the inoculation and replication processes can be carried out simultaneously. Therefore, the relatively fast rate of generating copies at the site inoculated by the first target molecule will effectively exclude the second target molecule from inoculating the site. In some cases, the inoculation of the target molecule will trigger a reaction that fills the site to capacity by a process other than replicating the target molecule. For example, the capture of the target molecule at the site can trigger a chain reaction, which ultimately prevents the site from capturing the second target molecule. A chain reaction can occur at a rate that exceeds the rate at which the target molecule is captured, thereby occurring under exclusion amplification conditions.

[0167] As exemplified by target nucleic acid, exclusion amplification can utilize the relatively slow rate of starting repeated reactions (such as chain reactions) at array sites when applied to other target molecules relative to the relatively fast rate of continuing recombination reactions once started. In the example of the preceding paragraph, exclusion amplification occurs due to the relatively slow target molecule inoculation rate (e.g., relatively slow diffusion) relative to the relatively fast rate of reacting, for example, filling sites with copies of target molecule seeds. In another exemplary embodiment, exclusion amplification can occur due to the formation delay (e.g., delay or slow activation) of the first copy of the target molecule inoculated site relative to the relatively fast rate of producing subsequent copies to fill sites. In this example, individual sites can be inoculated with several different target molecules. However, the formation of the first copy of any given target molecule can be randomly activated, so that the average rate of the first copy formation is relatively slow compared to the rate of producing subsequent copies. In this case, although individual sites can be inoculated with several different target molecules, exclusion amplification will only allow replication of one of those target molecules.

[0168] Thus, the present disclosure provides a method for preparing a molecular array, which may include the following steps: (a) providing reagents comprising (i) an array of sites, and (ii) a solution having a plurality of different target molecules, wherein the number of target molecules in the solution exceeds the number of sites in the array, wherein the different target molecules have fluid pathways to the plurality of sites, and wherein each site includes a capacity for several of the plurality of different target molecules; and (b) reacting the reagents to produce a plurality of sites, each of which has a single target molecule from the plurality, or to produce a plurality of sites, each of which has a population of pure copies of an individual target molecule from the solution, wherein the reaction comprises simultaneously (i) transporting the different molecules to the sites at an average transport rate, and (ii) initiating a reaction to fill the sites to capacity at an average reaction rate, wherein the average reaction rate exceeds the average transport rate. In some embodiments, step (b) may alternatively be performed by reacting the reagents to produce a plurality of sites each having a single target molecule from the plurality, or to produce a plurality of sites each having a population of pure copies of individual target molecules from a solution, wherein the reaction comprises (i) initiating a repeated reaction (such as a chain reaction) at each site to form a product from the target molecule, and (ii) continuing the reaction at each site to form subsequent products, wherein the average rate at which the reaction occurs at the site exceeds the average rate at which the reaction is initiated at the site.

[0169] In the above-mentioned non-nucleic acid embodiment, the target molecule can be an initiator that undergoes repeated reactions at each site of the array. For example, repeated reactions can form polymers that exclude other target molecules from occupying the site. Alternatively, repeated reactions can form one or more polymers that constitute molecular copies of the target molecule that is transported to the site.

[0170] Exemplary embodiments

[0171] Embodiment 1. A method for amplifying a nucleic acid, comprising:

[0172] (a) providing an amplification reagent comprising:

[0173] (i) an array of amplification sites,

[0174] wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence,

[0175] wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, and

[0176] (ii) a solution comprising a plurality of different modified double-stranded target nucleic acids,

[0177] wherein the different modified target nucleic acid comprises a first universal capture binding sequence at the 3' end that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity to the first capture sequence; and

[0178] (b) reacting the amplification reagents to generate a plurality of amplification sites, each of which comprises a clonal population of amplicons of a respective target nucleic acid from the solution.

[0179] Embodiment 2. A method for amplifying a nucleic acid, comprising:

[0180] (a) providing an amplification reagent comprising:

[0181] (i) an array of amplification sites,

[0182] wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence,

[0183] wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, and

[0184] (ii) a solution comprising a plurality of different modified target nucleic acids,

[0185] wherein the different modified target nucleic acid comprises a first universal capture binding sequence at the 3' end that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity to the first capture sequence; and

[0186] (b) reacting the amplification reagents to produce a plurality of amplification sites, each of the amplification sites comprising a clonal population of amplicons of a respective target nucleic acid from the solution, wherein the reacting comprises:

[0187] (i) generating first amplicons from individual target nucleic acids transported to each of said amplification sites, and

[0188] (ii) generating subsequent amplicons from said individual target nucleic acids transported to each of said amplification sites or from said first amplicon,

[0189] Wherein an average rate at which the subsequent amplicons are produced at the amplification site is less than an average rate at which the first amplicons are produced at the amplification site.

[0190] Embodiment 3. A method for determining a nucleic acid sequence, comprising performing a sequencing procedure that detects an apparent clonal population of amplicons at each of a plurality of amplicon sites on an array, wherein the array is prepared by a method comprising:

[0191] (a) providing an amplification reagent comprising:

[0192] (i) multiple amplification sites,

[0193] wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence,

[0194] wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, and

[0195] (ii) a solution comprising a plurality of different modified target nucleic acids,

[0196] wherein the different modified target nucleic acid comprises a first universal capture binding sequence at the 3' end that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity with the first capture sequence;

[0197] (b) reacting the amplification reagent.

[0198] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the number of the different modified target nucleic acids in the solution exceeds the number of amplification sites in the array,

[0199] wherein the different modified target nucleic acids have fluidic access to the plurality of amplification sites, and

[0200] Each of the amplification sites contains the capacity for several of the multiple different nucleic acids.

[0201] Embodiment 5. The method of any one of Embodiments 1 to 4, wherein the reaction comprises simultaneously:

[0202] (i) transporting the different modified target nucleic acids to the amplification site at an average transport rate, and

[0203] (ii) amplifying the target nucleic acid located at the amplification site at an average amplification rate, wherein the average amplification rate is less than the average transport rate.

[0204] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the plurality of different modified target nucleic acids in the solution are at concentrations that simultaneously result in:

[0205] (i) transporting the different modified target nucleic acids from the solution to the amplification site, and

[0206] (ii) amplifying the target nucleic acid located at the amplification site at an amplification rate to produce an array of amplicon sites, each of which comprises an apparent clonal population of the amplicon.

[0207] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the first universal capture binding sequence has less than 100% complementarity with the first capture sequence.

[0208] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the first universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the first capture sequence.

[0209] Embodiment 9. A method according to any one of embodiments 1 to 8, wherein the different modified target nucleic acids comprise a heterogeneous population of first universal capture binding sequences, wherein the heterogeneous population comprises individual first universal capture binding sequences having (i) 1, 2 or 3 nucleotides that are not complementary to the first capture sequence, or (ii) 100% complementarity to the first capture sequence.

[0210] Embodiment 10. The method of any one of embodiments 1 to 9, wherein members of the heterogeneous population having 100% complementarity to the first capture sequence are present in greater numbers than other members of the heterogeneous population.

[0211] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the first universal capture binding sequence has a length that is smaller than the length of the first capture sequence.

[0212] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the first universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

[0213] Embodiment 13. A method according to any one of embodiments 1 to 12, wherein the different modified target nucleic acids comprise a heterogeneous population of first universal capture binding sequences, wherein the heterogeneous population comprises individual first universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

[0214] Embodiment 14. The method of any one of Embodiments 1 to 13, wherein the heterogeneous population further comprises individual first universal capture binding sequences having (iii) a length that is smaller than a length of the first capture sequence.

[0215] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the individual first universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

[0216] Embodiment 16. The method of any one of embodiments 1 to 15, wherein individual members of the heterogeneous population having a length smaller than the length of the first capture sequence contain 1, 2 or 3 nucleotides that are not complementary to the sequence of the first capture sequence, or are 100% complementary to the sequence of the first capture sequence.

[0217] Embodiment 17. A method according to any one of embodiments 1 to 16, wherein the different modified target nucleic acid comprises at the 5' end a second universal capture binding sequence having a complement that has a lower affinity for the second capture sequence than a second universal capture binding sequence having a complement that is 100% complementary to the second capture sequence.

[0218] Embodiment 18. The method of any one of Embodiments 1 to 17, wherein the complement of the second universal capture binding sequence has less than 100% complementarity with the second capture sequence.

[0219] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the complement of the second universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the second capture sequence.

[0220] Embodiment 20. A method according to any one of embodiments 1 to 19, wherein the different modified target nucleic acids comprise a heterogeneous population of a second universal capture binding sequence, wherein the heterogeneous population comprises individual second universal capture binding sequences, the second universal capture sequence comprising a complement having (i) 1, 2 or 3 nucleotides that are not complementary to the second capture sequence, or (ii) 100% complementarity to the second capture sequence.

[0221] Embodiment 21. The method of any one of embodiments 1 to 20, wherein members of the heterogeneous population comprising a complement having 100% complementarity to the second capture sequence are present in greater numbers than other members of the heterogeneous population.

[0222] Embodiment 22. The method of any one of Embodiments 1 to 21, wherein the second universal capture binding sequence has a length that is smaller than the length of the second capture sequence.

[0223] Embodiment 23. The method of any one of Embodiments 1 to 22, wherein the second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0224] Embodiment 24. A method according to any one of embodiments 1 to 23, wherein the different modified target nucleic acids comprise a heterogeneous population of second universal capture binding sequences, wherein the heterogeneous population comprises individual second universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0225] Embodiment 25. The method of any one of Embodiments 1 to 24, wherein the heterogeneous population further comprises an individual second universal capture binding sequence having (iii) a length that is smaller than the length of the second capture sequence.

[0226] Embodiment 26. The method of any one of Embodiments 1 to 25, wherein the individual second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0227] Embodiment 27. A method according to any one of embodiments 1 to 26, wherein individual members of the heterogeneous population having a length smaller than the length of the second capture sequence comprise complements comprising 1, 2 or 3 nucleotides that are not complementary to the sequence of the second capture sequence, or 100% complementarity to the sequence of the second capture sequence.

[0228] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the target nucleic acid is DNA.

[0229] Embodiment 29. The method of any one of Embodiments 1 to 28, wherein the array of amplification sites comprises an array of features on a surface.

[0230] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the area of ​​each of the features is larger than the diameter of the exclusion volume of the target nucleic acid transported to the amplification site.

[0231] Embodiment 31. The method of any one of Embodiments 1 to 30, wherein the features are discontinuous and separated by interstitial regions of the surface lacking capture agents.

[0232] Embodiment 32. The method of any one of Embodiments 1 to 31, wherein each of the features comprises a bead, a hole, a channel, a ridge, a protrusion, or a combination thereof.

[0233] Embodiment 33. The method of any one of Embodiments 1 to 2, wherein the array of amplification sites comprises beads in solution or beads on a surface.

[0234] Embodiment 34. The method of any one of embodiments 1 to 3, wherein amplification of the target nucleic acid transported to the amplification site occurs isothermally.

[0235] Embodiment 35. The method of any one of embodiments 1 to 34, wherein amplification of the different modified target nucleic acids transported to the amplification site does not include a denaturation cycle.

[0236] Embodiment 36. The method of any one of embodiments 1 to 35, wherein the plurality of amplification sites of the clonal population comprising amplicons exceeds 40% of the amplification sites through which the different modified target nucleic acids had fluidic access during (b).

[0237] Embodiment 37. The method of any one of embodiments 1 to 36, wherein during (b) a sufficient number of amplicons are generated from the individual target nucleic acids at the individual amplification sites, respectively, to fill the capacity of the corresponding amplification sites.

[0238] Embodiment 38. The method of any one of embodiments 1 to 37, wherein the rate at which the amplicons are produced to fill the capacity of the corresponding amplification sites is less than the rate at which the individual target nucleic acids are transported to the individual amplification sites, respectively.

[0239] Embodiment 39. The method of any one of embodiments 1 to 38, wherein the transport comprises passive diffusion.

[0240] Embodiment 40. The method of any one of embodiments 1 to 39, wherein the amplification reagents further comprise a polymerase and a recombinase.

[0241] Embodiment 41. A method for generating a library, comprising:

[0242] providing a solution of a plurality of double-stranded target nucleic acids;

[0243] ligating universal adaptors to both ends of the double-stranded target nucleic acid to form a first plurality of modified target nucleic acids,

[0244] wherein each of said modified target nucleic acids comprises a target nucleic acid flanked by said universal adaptors,

[0245] wherein the universal adaptor comprises (i) a double-stranded nucleic acid region, and (ii) a single-stranded non-complementary nucleic acid region comprising a universal capture binding sequence at the 3' end,

[0246] wherein the universal capture binding sequences comprise a heterogeneous population, and

[0247] wherein the ligating covalently attaches the double-stranded nucleic acid region of the universal adaptor to each end of the double-stranded target fragment.

[0248] Embodiment 42. The method of embodiment 41, wherein members of the heterogeneous population of universal capture binding sequences at the 3' end differ from each other by 1, 2 or 3 nucleotides.

[0249] Embodiment 43. The method of embodiment 41 or 42, wherein members of the heterogeneous population of universal capture binding sequences at the 3' end have lengths that differ from each other by 1 to 12 nucleotides.

[0250] Embodiment 44. The method of any one of embodiments 41 to 43, wherein members of the heterogeneous population of universal capture binding sequences at the 3' end differ from each other by 1, 2 or 3 nucleotides, differ from each other by 1 to 12 nucleotides, or a combination thereof.

[0251] Embodiment 45. The method of any one of Embodiments 41 to 44, wherein the single-stranded non-complementary nucleic acid region comprises a second universal capture binding sequence at the 5' end.

[0252] Embodiment 46. The method of any one of embodiments 41 to 45, wherein members of the heterogeneous population of said second universal capture binding sequences at the 5' end differ from each other by 1, 2 or 3 nucleotides.

[0253] Embodiment 47. The method of any one of Embodiments 41 to 46, wherein members of the heterogeneous population of said second universal capture binding sequences at the 5' end have lengths that differ from each other by 1 to 12 nucleotides.

[0254] Embodiment 48. The method of any one of embodiments 41 to 47, wherein members of the heterogeneous population of said second universal capture binding sequences at the 5' end differ from each other by 1, 2 or 3 nucleotides, differ from each other by 1 to 12 nucleotides, or a combination thereof.

[0255] Embodiment 49. A composition comprising an array of amplification sites and at least one target nucleic acid bound to the amplification sites,

[0256] wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence,

[0257] wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence,

[0258] wherein the target nucleic acid comprises at the 3' end a first universal capture binding sequence that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity with the first capture sequence,

[0259] Wherein the target nucleic acid universal capture binding sequence hybridizes to the first capture sequence.

[0260] Embodiment 50. The composition of embodiment 49, wherein the first universal capture binding sequence has less than 100% complementarity with the first capture sequence.

[0261] Embodiment 51. The composition of embodiment 49 or 50, wherein the first universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the first capture sequence.

[0262] Embodiment 52. The composition of any one of Embodiments 49 to 51, wherein at least 30% of the amplification sites of the array are occupied by at least one target nucleic acid.

[0263] Embodiment 53. A composition of any one of embodiments 49 to 52, wherein the first universal capture binding sequence comprises a heterogeneous population, wherein the heterogeneous population comprises individual first universal capture binding sequences, the first universal capture binding sequence having (i) 1, 2 or 3 nucleotides that are not complementary to the first capture sequence, or (ii) 100% complementarity to the first capture sequence, and wherein members of the heterogeneous population bind to different amplification sites.

[0264] Embodiment 54. The composition of any one of Embodiments 49 to 53, wherein members of the heterogeneous population having 100% complementarity to the first capture sequence are present in greater numbers than other members of the heterogeneous population.

[0265] Embodiment 55. The method of any one of Embodiments 49 to 54, wherein the second universal capture binding sequence has a length that is smaller than the length of the second capture sequence.

[0266] Embodiment 56. The composition of any one of Embodiments 49 to 55, wherein the second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0267] Embodiment 57. A composition of any one of Embodiments 49 to 56, wherein the composition comprises a plurality of different target nucleic acids, the different target nucleic acids comprising a heterogeneous population of first universal capture binding sequences, wherein the heterogeneous population comprises individual first universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

[0268] Embodiment 58. The composition of any one of Embodiments 49 to 57, wherein the heterogeneous population further comprises individual first universal capture binding sequences having (iii) a length that is less than a length of the first capture sequence.

[0269] Embodiment 59. The composition of any one of embodiments 49 to 58, wherein the individual first universal capture binding sequences comprising a shortened length have a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

[0270] Embodiment 60. The composition of any one of Embodiments 49 to 59, wherein individual members of the heterogeneous population having shortened length comprise 1, 2 or 3 nucleotides that are not complementary to the sequence of the first capture sequence, or 100% complementarity to the sequence of the first capture sequence.

[0271] Embodiment 61. A composition of any one of Embodiments 49 to 60, wherein the composition comprises a plurality of different target nucleic acids, wherein the different target nucleic acids comprise at the 5' end a second universal capture binding sequence having a complement that has a lower affinity for the second capture sequence than a second universal capture binding sequence having a complement that is 100% complementary to the second capture sequence.

[0272] Embodiment 62. The composition of any one of Embodiments 49 to 61, wherein the complement of the second universal capture binding sequence has less than 100% complementarity with the second capture sequence.

[0273] Embodiment 63. The composition of any one of Embodiments 49 to 62, wherein the complement of the second universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the second capture sequence.

[0274] Embodiment 64. A composition of any one of embodiments 49 to 63, wherein the composition comprises a plurality of different target nucleic acids, the different target nucleic acids comprising a heterogeneous population of a second universal capture binding sequence, wherein the heterogeneous population comprises individual second universal capture binding sequences, the second universal capture binding sequence comprising a complement having (i) 1, 2 or 3 nucleotides that are not complementary to the second capture sequence, or (ii) 100% complementarity to the second capture sequence.

[0275] Embodiment 65. The composition of any one of Embodiments 49 to 64, wherein members of the heterogeneous population comprising a complement having 100% complementarity to the second capture sequence are present in greater numbers than other members of the heterogeneous population.

[0276] Embodiment 66. The composition of any one of Embodiments 49 to 65, wherein the target nucleic acid comprises a second universal capture binding sequence at the 5' end, the second universal capture binding sequence having a length that is smaller than the length of the second capture sequence.

[0277] Embodiment 67. The composition of any one of Embodiments 49 to 66, wherein the second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0278] Embodiment 68. A composition of any one of Embodiments 49 to 67, wherein the composition comprises a plurality of different target nucleic acids, the different target nucleic acids comprising a heterogeneous population of second universal capture binding sequences, wherein the heterogeneous population comprises individual second universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0279] Embodiment 69. The composition of any one of Embodiments 49 to 68, wherein the heterogeneous population further comprises an individual second universal capture binding sequence having (iii) a length that is less than the length of the second capture sequence.

[0280] Embodiment 70. The composition of any one of embodiments 49 to 69, wherein the individual second universal capture binding sequences have a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

[0281] Embodiment 71. A composition of any one of Embodiments 49 to 70, wherein individual members of the heterogeneous population having a length less than the length of the second capture sequence comprise complements comprising 1, 2 or 3 nucleotides that are not complementary to the sequence of the second capture sequence, or 100% complementarity to the sequence of the second capture sequence.

[0282] Embodiment 72. The composition of any one of Embodiments 49 to 71, wherein the target nucleic acid is DNA.

[0283] Example

[0284] The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0285] Example 1

[0286] General assay methods and conditions

[0287] Unless otherwise stated, this describes the general assay conditions used in the examples described herein.

[0288] Nextera TM Library preparation begins with the generation of a nucleic acid library by introducing the universal portion of the adapters through tagmentation of human gDNA. This universal tag fragmentation is then divided into individual reactions, one for each different adapter pair (PCR1-PCR21). In each of these reactions, Nextera TMThe modified adapters were introduced by replacing the standard P5 / P7 adapters with modified adapters using XT library preparation reagents (Illumina, San Diego, California) through 12 PCR cycles. The modified adapters were designed with variations from the standard P5 / P7 sequences (outlined below) and synthesized by Integrated DNA Technologies (IDT, Skokie, Illinois).

[0289] Example 2

[0290] Evaluation of kinetic delays for adapter mutants

[0291] A series of modified adapters were used to generate the same TM The libraries (Table 1) were different. These adapters were slightly shorter than the standards (-4 bp or -9 bp) or had 1, 2 or 3 mismatches introduced along the length of the P5 / P7 region ("wobble" bases: 1W, 2W or 3W, respectively). The mutations ranged from perfect P5 & P7 sequence (PCR1) to 3 mismatches at both ends (PCR 21).

[0292] The concentration of each library was then quantified and the libraries were normalized to the same concentration. Libraries with different modified adapters were then amplified separately using the KAPA Library Quantification Kit (Kapa Biosystems) for the Illumina platform with custom primers on a qPCR instrument (BioRad CFX384 Real-Time System) to simulate the conditions of the flow cell to generate the results in Table 1. The efficiency was calculated based on the number of cycles required to reach the Ct (threshold cycle) defined by the qPCR standard.

[0293] Table 1.

[0294]

[0295]

[0296] αβ-4 refers to four base pairs removed from the adaptor; αβ-9 refers to nine base pairs removed from the adaptor; 1W, 2W, 3W refer to 1, 2 or 3 wobble mismatches, respectively, present along the length of the region that binds to the capture nucleic acid present on the array surface; 1MM, true mismatch; FCP5, FCP7, refer to full-length P5 and P7 primers, respectively.

[0297] Example 3

[0298] Evaluation of kinetic delays in adapter mutants using sequencing

[0299] Different mutant adapters were selected and analyzed individually or in groups on HiSeq TM X flow cell (Table 2). Illumina HiSeq was run using a standard kit TM All sequencing was performed on an X-ray machine.

[0300] Table 2

[0301]

[0302]

[0303] result

[0304] like Figure 3A As shown, lanes 1, 2 and 4 are reactions performed using adapters with fewer mismatches and higher efficiency, and result in high intensity and a high percentage of clusters through the filter as expected. Lanes 3 and 5 are reactions performed using adapters with more mismatches and lower efficiency, resulting in low intensity and a low percentage of clusters through the filter as expected. Lanes 6, 7 and 8 are mixing of adapters with high efficiency and low efficiency. Counterintuitively, the mixture does not perform as well as the average value (e.g., between high efficiency and low efficiency) of the performance of individual components, but is superior to all single types of libraries in both intensity and clusters through the filter. Therefore, the surprising result is that by reducing the average homology, even if the average rate of amplification is reduced, the rate of the so-called monoclonality of the nanopore is improved. The novelty is that a certain degree of variability is introduced into the adapter sequence, so that there is now a certain efficiency range in the template population. In this way, when multiple templates are inoculated into the cushion, there is usually a template that has an advantage over all other templates, making it obviously dominant on the cushion. Furthermore, the reduced homology is corrected in daughter copies, so that the delay is only introduced into the first copy without affecting the efficiency of subsequent amplifications.

[0305] Select different mutant adapters and perform sequencing on HiSeq individually or in groups. TM X flow cell (Table 3). As described above, standard kits were used on Illumina HiSeq T All sequencing was performed on an MX instrument.

[0306] Table 3

[0307] Flow cell lanes Adapters used in lanes Lane 1 PCR 1 Lane 2 PCR 3 Lane 3 PCR 10 Lane 4 PCR 16 Lane 5 PCR 1,3,6,8,9,10,16-18 Lane 6 PCR 21 Lane 7 PCR 1,3,6,8,9,10,16-18 Lane 8 PCR 1,3,10

[0308] When a set of mutant adapters are run in a mixture (such as lane 7), they are combined at equal concentrations. In a conventional sequencing run, i.e., without using the proposed method, a mixed library of identical adapters at the same concentration would result in equal read ratios on the flow cell. Figure 3BAs shown, a mixture of different mutant adaptors yields a representation of the final read counts that is proportional to their efficiency rather than their seeding concentration, thus demonstrating the efficacy of the proposed approach, i.e., adaptors with lower affinity have longer kinetic delays, which results in a lower proportion of final reads.

[0309] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions in, for example, GenBank and RefSeq and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions from GenBank and RefSeq) are incorporated by reference in their entirety. Supplementary materials (e.g., supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) cited in publications are also incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein by reference, the disclosure of the present application shall prevail. The foregoing detailed description and examples are given only for the purpose of clear understanding. No unnecessary limitations should be understood from them. The present disclosure is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art will be included in the present disclosure defined by the claims.

[0310] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and the like used in the specification and claims are to be understood in all instances as modified by the term "about". Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. Regardless, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0311] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a certain range which necessarily results from the standard deviation found in their respective testing measurements.

[0312] Unless otherwise indicated, all headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading.

Claims

1. A method for amplifying a nucleic acid, comprising: (a) providing an amplification reagent comprising: (i) an array of amplification sites, wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence, wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, and (ii) a solution comprising a plurality of different modified double-stranded target nucleic acids, wherein the different modified target nucleic acid comprises a first universal capture binding sequence at the 3' end that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity with the first capture sequence; and (b) reacting the amplification reagents to generate a plurality of amplification sites, each of the amplification sites comprising a clonal population of amplicons of a respective target nucleic acid from the solution.

2. A method for amplifying a nucleic acid, comprising: (a) providing an amplification reagent comprising: (i) an array of amplification sites, wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence, wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, and (ii) a solution comprising a plurality of different modified target nucleic acids, wherein the different modified target nucleic acid comprises a first universal capture binding sequence at the 3' end that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity with the first capture sequence; and (b) reacting the amplification reagents to produce a plurality of amplification sites, each of the amplification sites comprising a clonal population of amplicons of a respective target nucleic acid from the solution, wherein the reacting comprises: (i) generating first amplicons from individual target nucleic acids transported to each of said amplification sites, and (ii) generating subsequent amplicons from said individual target nucleic acids transported to each of said amplification sites or from said first amplicon, Wherein an average rate at which the subsequent amplicons are produced at the amplification sites is greater than an average rate at which the first amplicons are produced at the amplification sites.

3. A method for determining a nucleic acid sequence comprising performing a sequencing procedure that detects an apparent clonal population of amplicons at each of a plurality of amplicon sites on an array, wherein the array is prepared by a method comprising: (a) providing an amplification reagent comprising: (i) multiple amplification sites, wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence, wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, and (ii) a solution comprising a plurality of different modified target nucleic acids, wherein the different modified target nucleic acid comprises a first universal capture binding sequence at the 3' end that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity with the first capture sequence; (b) reacting the amplification reagent.

4. The method of claim 1, 2 or 3, wherein the number of different modified target nucleic acids in the solution exceeds the number of amplification sites in the array, wherein the different modified target nucleic acids have fluidic access to the plurality of amplification sites, and Each of said amplification sites contains a capacity for several nucleic acids of said plurality of different modified target nucleic acids.

5. The method of claim 1, wherein the reacting comprises simultaneously: (i) transporting the different modified target nucleic acids to the amplification site at an average transport rate, and (ii) amplifying the target nucleic acid located at the amplification site at an average amplification rate, wherein the average amplification rate is greater than the average transport rate.

6. The method of claim 3, wherein the concentrations of the plurality of different modified target nucleic acids in the solution simultaneously result in: (i) transporting the different modified target nucleic acids from the solution to the amplification site, and (ii) amplifying the target nucleic acid located at the amplification site at an amplification rate to produce an array of amplicon sites, each of which comprises an apparent clonal population of the amplicon.

7. The method of any one of claims 1 to 3, wherein the first universal capture binding sequence has less than 100% complementarity with the first capture sequence.

8. The method of claim 7, wherein the first universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the first capture sequence.

9. The method of claim 7, wherein the different modified target nucleic acids comprise a heterogeneous population of first universal capture binding sequences, wherein the heterogeneous population comprises individual first universal capture binding sequences, wherein the first universal capture binding sequences have (i) 1, 2 or 3 nucleotides that are not complementary to the first capture sequence, or (ii) 100% complementarity to the first capture sequence.

10. The method of claim 9, wherein members of the heterogeneous population having 100% complementarity to the first capture sequence are present in greater numbers than other members of the heterogeneous population.

11. The method of any one of claims 1 to 3, wherein the first universal capture binding sequence has a length that is smaller than a length of the first capture sequence.

12. The method of claim 11, wherein the first universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

13. The method of claim 11, wherein the different modified target nucleic acids comprise a heterogeneous population of first universal capture binding sequences, wherein the heterogeneous population comprises individual first universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

14. The method of claim 9, wherein the heterogeneous population further comprises individual first universal capture binding sequences having (iii) a length that is less than a length of the first capture sequence.

15. The method of claim 14, wherein the individual first universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

16. The method of claim 14, wherein individual members of the heterogeneous population having a length less than the length of the first capture sequence comprise 1, 2 or 3 nucleotides that are not complementary to the sequence of the first capture sequence, or are 100% complementary to the sequence of the first capture sequence.

17. The method of any one of claims 1 to 3, wherein the different modified target nucleic acid comprises at the 5' end a second universal capture binding sequence having a complement that has a lower affinity for the second capture sequence than a second universal capture binding sequence having a complement that is 100% complementary to the second capture sequence.

18. The method of claim 17, wherein the complement of the second universal capture binding sequence has less than 100% complementarity with the second capture sequence.

19. The method of claim 18, wherein the complement of the second universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the second capture sequence.

20. The method of claim 18, wherein the different modified target nucleic acids comprise a heterogeneous population of a second universal capture binding sequence, wherein the heterogeneous population comprises individual second universal capture binding sequences, the second universal capture binding sequences comprising complements having (i) 1, 2 or 3 nucleotides that are not complementary to the second capture sequence, or (ii) 100% complementarity to the second capture sequence.

21. The method of claim 20, wherein members of the heterogeneous population comprising a complement having 100% complementarity to the second capture sequence are present in greater numbers than other members of the heterogeneous population.

22. The method of any one of claims 1 to 3, wherein the target nucleic acid comprises a second universal capture binding sequence at the 5' end, wherein the second universal capture binding sequence has a length that is smaller than the length of the second capture sequence.

23. The method of claim 22, wherein the second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

24. The method of claim 22, wherein the different modified target nucleic acids comprise a heterogeneous population of second universal capture binding sequences, wherein the heterogeneous population comprises individual second universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

25. The method of claim 20, wherein the heterogeneous population further comprises an individual second universal capture binding sequence having (iii) a length that is less than a length of the second capture sequence.

26. The method of claim 25, wherein the individual second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

27. The method of claim 26, wherein individual members of the heterogeneous population having a length less than the length of the second capture sequence comprise complements comprising 1, 2 or 3 nucleotides that are not complementary to the sequence of the second capture sequence, or 100% complementarity to the sequence of the second capture sequence.

28. The method of any one of claims 1 to 3, wherein the target nucleic acid is DNA.

29. The method of any one of claims 1 to 3, wherein the array of amplification sites comprises an array of features on a surface.

30. The method of claim 29, wherein the area of ​​each of said features is larger than the diameter of an exclusion volume of said target nucleic acid transported to said amplification site.

31. The method of claim 30, wherein said features are discontinuous and separated by interstitial regions of said surface lacking capture agent.

32. The method of claim 29, wherein each of said features comprises a bead, a hole, a channel, a protrusion, or a combination thereof.

33. The method of claim 32, wherein each protrusion is a ridge.

34. The method of any one of claims 1 to 3, wherein the array of amplification sites comprises beads in solution or beads on a surface.

35. The method of any one of claims 1 to 3, wherein amplification of the target nucleic acid transported to the amplification site occurs isothermally.

36. The method of any one of claims 1 to 3, wherein amplification of the different modified target nucleic acids transported to the amplification site does not include a denaturation cycle.

37. The method of any one of claims 1 to 3, wherein the plurality of amplification sites of the clonal population comprising amplicons exceeds 40% of the amplification sites to which the different modified target nucleic acids had fluidic access during (b).

38. The method of any one of claims 1 to 3, wherein during (b) a sufficient number of amplicons are generated from the individual target nucleic acids at the individual amplification sites to fill the capacity of the corresponding amplification sites.

39. The method of claim 38, wherein the rate at which the amplicons are produced to fill the capacity of the corresponding amplification sites is greater than the rate at which the individual target nucleic acids are transported to the individual amplification sites, respectively.

40. The method of any one of claims 1 to 3, wherein the transport comprises passive diffusion.

41. The method of any one of claims 1 to 3, wherein the amplification reagents further comprise a polymerase and a recombinase.

42. A composition comprising an array of amplification sites and at least one target nucleic acid bound to the amplification sites, wherein the amplification site comprises two capture nucleic acid populations, each population comprising a capture sequence, wherein the first population comprises a first capture sequence and the second population comprises a second capture sequence, wherein the target nucleic acid comprises at the 3' end a first universal capture binding sequence that has a lower affinity for the first capture sequence than a first universal capture binding sequence that has 100% complementarity with the first capture sequence, Wherein the target nucleic acid universal capture binding sequence hybridizes to the first capture sequence.

43. The composition of claim 42, wherein the first universal capture binding sequence has less than 100% complementarity with the first capture sequence.

44. The composition of claim 42, wherein the first universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the first capture sequence.

45. The composition of claim 42, wherein at least 30% of the amplification sites of the array are occupied by at least one target nucleic acid.

46. ​​The composition of claim 45, wherein the first universal capture binding sequence comprises a heterogeneous population, wherein the heterogeneous population comprises individual first universal capture binding sequences, the first universal capture binding sequence having (i) 1, 2 or 3 nucleotides that are not complementary to the first capture sequence, or (ii) 100% complementarity to the first capture sequence, and wherein members of the heterogeneous population bind to different amplification sites.

47. The composition of claim 46, wherein members of the heterogeneous population having 100% complementarity to the first capture sequence are present in greater numbers than other members of the heterogeneous population.

48. The method of claim 42, wherein the target nucleic acid comprises a second universal capture binding sequence at the 5' end, wherein the second universal capture binding sequence has a length that is less than the length of the second capture sequence.

49. The composition of claim 48, wherein the second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

50. The composition of claim 49, wherein the composition comprises a plurality of different target nucleic acids, the different target nucleic acids comprising a heterogeneous population of first universal capture binding sequences, wherein the heterogeneous population comprises individual first universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

51. The composition of claim 46, wherein the heterogeneous population further comprises individual first universal capture binding sequences having (iii) a length that is less than a length of the first capture sequence.

52. The composition of claim 51, wherein the individual first universal capture binding sequences comprising a shortened length have a length that is 1 to 12 nucleotides less than the length of the first capture sequence.

53. The composition of claim 51, wherein individual members of the heterogeneous population having shortened length comprise 1, 2, or 3 nucleotides that are not complementary to the sequence of the first capture sequence, or 100% complementarity to the sequence of the first capture sequence.

54. The composition of claim 42, wherein the composition comprises a plurality of different target nucleic acids, wherein the different target nucleic acids comprise at the 5' end a second universal capture binding sequence having a complement that has a lower affinity for the second capture sequence than a second universal capture binding sequence having a complement that is 100% complementary to the second capture sequence.

55. The composition of claim 54, wherein the complement of the second universal capture binding sequence has less than 100% complementarity with the second capture sequence.

56. The composition of claim 55, wherein the complement of the second universal capture binding sequence comprises 1, 2 or 3 nucleotides that are not complementary to the second capture sequence.

57. The composition of claim 55, wherein the composition comprises a plurality of different target nucleic acids, the different target nucleic acids comprising a heterogeneous population of a second universal capture binding sequence, wherein the heterogeneous population comprises individual second universal capture binding sequences, the second universal capture binding sequence comprising a complement having (i) 1, 2 or 3 nucleotides that are not complementary to the second capture sequence, or (ii) 100% complementarity to the second capture sequence.

58. The composition of claim 57, wherein members of the heterogeneous population comprising a complement having 100% complementarity to the second capture sequence are present in greater numbers than other members of the heterogeneous population.

59. The composition of claim 42, wherein the target nucleic acid comprises a second universal capture binding sequence at the 5' end, the second universal capture binding sequence having a length that is less than the length of the second capture sequence.

60. The composition of claim 59, wherein the second universal capture binding sequence has a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

61. The composition of claim 59, wherein the composition comprises a plurality of different target nucleic acids, the different target nucleic acids comprising a heterogeneous population of second universal capture binding sequences, wherein the heterogeneous population comprises individual second universal capture binding sequences having a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

62. The composition of claim 57, wherein the heterogeneous population further comprises an individual second universal capture binding sequence having (iii) a length that is less than a length of the second capture sequence.

63. The composition of claim 62, wherein the individual second universal capture binding sequences have a length that is 1 to 12 nucleotides less than the length of the second capture sequence.

64. The composition of claim 63, wherein individual members of the heterogeneous population having a length less than the length of the second capture sequence comprise complements comprising 1, 2 or 3 nucleotides that are not complementary to the sequence of the second capture sequence, or 100% complementarity to the sequence of the second capture sequence.

65. The composition of claim 42, wherein the target nucleic acid is DNA.

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