Methods and compositions for manipulating nucleic acids

Abstract

The present disclosure provides methods, compositions, kits and systems for manipulating nucleic acids, the methods comprising isothermal amplification of nucleic acid templates using pre-implanted solid supports, such as recombinase-polymerase amplification (RPA). A fast and effective method for generating template nucleic acid molecules including specific nucleotide sequences bound to a solid support is provided. Such methods can be used, for example, to manipulate nucleic acids in preparation for analytical methods utilizing monoclonal nucleic acid populations.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 16 / 403,339, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application is hereby incorporated by reference into the electronic sequence listing materials filed at the same time. The materials in the electronic sequence listing are submitted in the form of a text (.txt) file titled "LT01179-1PCT_ST25.txt" created on April 29, 2020, with a file size of 1.67KB and is incorporated herein by reference in its entirety.

[0005] Incorporated by Reference

[0006] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Technical Field

[0007] The present disclosure generally relates to methods for manipulating and analyzing nucleic acids and compositions and kits for performing the same. Background Art

[0008] Manipulation of nucleic acid samples, such as nucleic acid amplification, is extremely useful in molecular biology and has wide applicability in virtually every aspect of biology, treatment, diagnosis, forensics, and research. Biological and medical research is increasingly turning to nucleic acid sequencing for enhancing biological research and medicine. For example, biologists and zoologists are turning to sequencing to study animal migration, species evolution, and the origin of traits. The medical community is using sequencing to study the cause of disease, sensitivity to drugs, and the cause of infection. The use of sequencing may be limited by the quantity and / or quality of the nucleic acid in the sample. Additionally, sequencing has historically been an expensive method, thus limiting its practice.

[0009] Generally, in order to increase the amount of nucleic acid that can be used for analysis, one or more primers are used to generate amplicons from nucleic acid molecules, wherein the amplicons are complementary to all or part of the template that generates them. Multiple amplification can also simplify the process and reduce overhead. For some downstream applications, monoclonal is desirable, because the different characteristics of the various nucleic acid molecules in the polyclonal population may complicate the interpretation of the determination data. In the example in which the monoclonal population of nucleic acid is expected to be used in the analytical method, there are also challenges in containing the monoclonal nucleic acid population and keeping it as a separation type and not containing or relatively not containing a large number of other nucleic acid impurities that are not the same as those nucleic acids in the monoclonal population. When attempting to analyze (e.g., sequencing) multiple samples of different nucleic acids in a high-throughput, automated, cost-effective manner, this is particularly a problem. In nucleic acid sequencing applications, the existence of a polyclonal population may complicate the interpretation of sequencing data; however, many sequencing systems are not sensitive enough to detecting the nucleotide sequence data from a single template nucleic acid molecule, so it is necessary to amplify the template nucleic acid molecule before sequencing.

[0010] An example of such amplification is recombinase-polymerase amplification (RPA), which is a DNA amplification method that uses an enzyme to bind an oligonucleotide primer to its complementary partner in a double-helix DNA, followed by isothermal amplification. Relative to traditional amplification methods, RPA provides a variety of advantages, including, for example, no need for initial thermal or chemical melting (melting), being able to operate at a low constant temperature without absolute temperature control, and the reaction mixture (e.g., lacking a target polynucleotide) can be stored under dry conditions. These advantages show that RPA is a powerful and convenient tool for amplifying nucleic acid molecules. However, attempts to use RPA to prepare template nucleic acid molecules before sequencing have resulted in undesired polyclonal populations and / or insufficient preparations. Therefore, there is still a need to produce improved template nucleic acid molecules for molecular characterization, such as improved methods for sequencing. Summary of the invention

[0011] In some embodiments, the present disclosure generally relates to methods of manipulating nucleic acids and systems, compositions, kits, and apparatuses for performing the methods.

[0012] In some embodiments, provided herein is a rapid and efficient method for producing nucleic acid molecules including specific nucleotide sequences. Such methods can be used, for example, in manipulating nucleic acids for analysis preparation in methods using monoclonal nucleic acid populations. Some embodiments of the clonal amplification methods disclosed herein for producing monoclonal nucleic acid populations start with constrained nucleic acid molecule templates. The method for constraining nucleic acid molecules provided herein comprises capturing a single nucleic acid molecule by combining specific nucleotide sequences common to different nucleic acids to be amplified and analyzed. In order to produce different nucleic acid molecules with common specific nucleotides that can be used for easy restriction, a method disclosed herein comprises (a) obtaining a nucleic acid molecule population, such as a double-stranded, adapter-containing DNA library, in which, with respect to one chain of the molecule, each molecule contains a first continuous nucleotide sequence at the 5' end of the molecule, a second continuous nucleotide sequence at the 3' end of the molecule, and a third nucleotide sequence positioned between the first continuous nucleotide sequence and the second continuous nucleotide sequence, wherein the first continuous nucleotide sequence is different from the second continuous nucleotide sequence, and in the nucleic acid molecule population, the first continuous nucleotide sequence of the nucleic acid molecule is substantially the same and the second continuous nucleotide sequence of the nucleic acid molecule is substantially the same. (b) subjecting the nucleic acid molecule population to a nucleic acid amplification cycle in the presence of a forward primer and a reverse primer, wherein the forward primer contains an oligonucleotide sequence substantially identical to the first nucleotide sequence, and the reverse primer contains an oligonucleotide sequence complementary to the subsequence at the 5' end of the second continuous nucleotide sequence and connected to a fourth nucleotide sequence that is not complementary to the second continuous nucleotide sequence at the 3' end of the sequence complementary to the subsequence; and (c) subjecting the product of the amplification cycle of step (b) to amplification cycles in the presence of the forward primer and the reverse primer to produce a plurality of different nucleic acid products, wherein only one of the products includes a nucleotide sequence complementary to the fourth nucleotide sequence. In some embodiments of this method, the reverse primer contains a nucleotide sequence complementary to the 5' end of the second continuous nucleotide sequence, but does not contain a nucleotide sequence complementary to the 3' end of the second continuous nucleotide sequence.

[0013] Other embodiments of the methods provided herein for producing different nucleic acid molecules having a common specific nucleotide sequence that can be used, for example, to facilitate restriction include: (a) obtaining a population of nucleic acid molecules, such as a double-stranded, adaptor-containing DNA library, in which, with respect to one strand of the molecule, each molecule contains, at the 5' end of the molecule, a first contiguous nucleotide sequence, at the 3' end of the molecule, and a third nucleotide sequence positioned between the first contiguous nucleotide sequence and the second contiguous nucleotide sequence, wherein the first contiguous nucleotide sequence is different from the second contiguous nucleotide sequence, and in the population of nucleic acid molecules, the first contiguous nucleotide sequence of the nucleic acid molecule is The nucleic acid molecules are described in claim 1, wherein the sequences are substantially identical and the second contiguous nucleotide sequences of the nucleic acid molecules are substantially identical; and (b) subjecting the population of nucleic acid molecules to two or more cycles of nucleic acid amplification in the presence of one or more forward primers and a reverse primer, wherein the one or more forward primers contain an oligonucleotide sequence substantially identical to the first nucleotide sequence, and the reverse primer is capped at the 3' end and contains an oligonucleotide sequence complementary to the second nucleotide sequence linked at the 5' end of the oligonucleotide sequence to a fourth nucleotide sequence that is not complementary to the second nucleotide sequence, to produce nucleic acid products, wherein substantially all of the products include a nucleotide sequence complementary to the fourth nucleotide sequence.

[0014] In some embodiments, methods for producing one or more templated carriers, such as solid carriers, are provided herein. Such methods include, for example: a) forming a templated reaction mixture by combining one or more pre-implanted carriers, nucleotides, a recombinase, and a polymerase, wherein the one or more pre-implanted solid carriers comprise a population of substantially identical first primers attached and have a substantially monoclonal template nucleic acid molecule attached thereto, wherein the one or more pre-implanted solid carriers are formed in a separate pre-implantation reaction, the separate pre-implantation reaction being performed prior to the templated reaction, wherein the substantially monoclonal template nucleic acid molecule comprises a proximal segment comprising the first primer, in some embodiments, the first primer does not comprise 100 or more identical nucleotides, wherein the proximal segment attaches the template nucleic acid segment to the pre-implanted solid carrier, and wherein the pre-implanted solid carrier further comprises a proximal segment attached to the pre-implanted solid carrier and not attached to the template nucleic acid molecule; The templated reaction mixture is a templated reaction mixture in which a first primer is connected to a nucleic acid molecule, wherein the templated reaction mixture further comprises a substantially identical soluble second primer population, and wherein the template nucleic acid molecule comprises a primer binding site of a second primer at or near the end opposite to the proximal segment; and b) a templated reaction is performed by adding cations to the templated reaction mixture and incubating the reaction mixture under isothermal conditions for at least 10 minutes to amplify the template nucleic acid molecule in the templated reaction to produce one or more templated solid carriers, wherein each of the templated solid carriers comprises at least 100,000 substantially monoclonal template nucleic acid molecules, and wherein when the templated reaction is started, the template nucleic acid molecule is not present in the reaction mixture in solution, thereby producing one or more templated solid carriers. In some embodiments, one or more templated solid carriers are used in a sequencing reaction to determine the sequence of the template nucleic acid molecule. In other embodiments, the templated solid carrier is a templated bead, and the sequencing comprises distributing the bead in the hole of the solid carrier before performing the sequencing reaction. In some embodiments, one or more templated solid carriers include: a first templated solid carrier, which is connected to a substantially monoclonal population of template nucleic acid molecules having a first sequence; and at least one other templated solid carrier, which is connected to a substantially monoclonal population of template nucleic acid molecules having a second sequence, wherein the sequence of the first connected template nucleic acid molecule is different from the sequence of the second connected template nucleic acid molecule. In other embodiments, the substantially monoclonal template nucleic acid molecule connected to each pre-implanted solid carrier includes at least 70% of all template nucleic acid molecules connected to each pre-implanted solid carrier. In some embodiments, the template reaction mixture includes a pre-implanted solid carrier population.In other embodiments, each pre-implanted solid carrier in the solid carrier colony has between 10 and 50,000 substantially monoclonal template nucleic acid molecules connected thereto, and the one or more solid carriers are beads. In certain embodiments, pre-implanted reaction mixture and / or templated reaction mixture further comprise recombinase auxiliaries. In other embodiments, recombinase auxiliaries are single-stranded binding proteins and / or recombinase load proteins. In certain embodiments, at a temperature between 35 ° C and 45 ° C, incubate templated reaction mixture and / or pre-implanted reaction mixture. In certain embodiments, incubate templated reaction mixture 10 to 60 minutes. In certain embodiments, substantially monoclonal template nucleic acid molecules present on templated solid carriers are at least 100 times of substantially monoclonal template nucleic acid molecules present on pre-implanted solid carriers.

[0015] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits, and devices for performing the methods, wherein the methods are for determining the sequence of a template nucleic acid molecule, and include: performing a preimplantation reaction and a subsequent template reaction. In some embodiments, the preimplantation reaction produces a plurality of preimplanted solid carriers, wherein a separate preimplanted carrier in the plurality of preimplanted solid carriers comprises a plurality of first primers connected to the solid carrier, wherein the plurality of first primers have substantially the same sequence, and wherein some of the plurality of first primers are engaged with the template nucleic acid molecule and some of the plurality of first primers are not engaged with the template nucleic acid molecule. In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits, and apparatus for determining the sequence of a template nucleic acid molecule, the methods comprising: a) generating a population of pre-implanted solid carriers comprising an identical first primer population attached, wherein the pre-implanted solid carriers are generated under pre-implantation conditions, and wherein each of the pre-implanted solid carriers has between 10 and 100,000 substantially monoclonal template nucleic acid molecules comprising the first primer attached thereto, and comprising an attached first primer that is attached to the pre-implanted solid carrier and does not bind to the template nucleic acid molecule; b) forming a templated reaction by combining the population of pre-implanted solid carriers, nucleotides, a recombinase, a polymerase, and an identical second primer population in solution that is not attached to any substrate; A mixture of template nucleic acid molecules, wherein the template nucleic acid molecule comprises a primer binding site of the second primer at or near the end opposite to the proximal section comprising the first primer; c) starting a template reaction by adding a cation to the template reaction mixture, wherein when the template reaction is started, the template nucleic acid molecule is not present in the reaction mixture in solution; d) incubating the started template reaction mixture under isothermal conditions for at least 10 minutes to amplify the template molecule in the template reaction to produce one or more template solid carriers, wherein the one or more template solid carriers contain at least 10 times the number of substantially monoclonal template nucleic acid molecules connected on the template solid carriers that are present on the pre-implanted solid carriers; and e) sequencing the template nucleic acid molecules on the one or more template solid carriers to thereby determine the sequence of the template nucleic acid molecules. In some embodiments, the template nucleic acid molecules comprise two or more template nucleic acid molecules with different sequences. In some embodiments, the substantially monoclonal template nucleic acid molecules connected to the pre-implanted solid carriers and / or connected to the template solid carriers comprise template nucleic acid molecules with two or more different sequences. In further embodiments, the substantially monoclonal template nucleic acid molecule attached to each pre-implanted solid support comprises at least 70% of all template nucleic acid molecules attached to each pre-implanted solid support.In some embodiments, the templated solid carrier is a templated bead, and the sequencing is included in the distribution of beads in the holes of the solid carrier before the sequencing reaction is performed. In some embodiments, each pre-implanted solid carrier of the solid carrier colony has between 10 and 50,000 substantially monoclonal template nucleic acid molecules connected thereto, and the one or more solid carriers are beads. In some embodiments, the pre-implanted reaction mixture and / or the templated reaction mixture further include a recombinase auxiliary protein. In other embodiments, the recombinase auxiliary protein is a single-stranded binding protein and / or a recombinase load protein. In some embodiments, at a temperature between 35 ° C and 45 ° C, the templated reaction mixture and / or the pre-implanted reaction mixture are incubated. In some embodiments, the templated reaction mixture is incubated for 10 to 60 minutes. In some embodiments, a pre-implanted solid carrier is produced using a first recombinase-polymerase amplification (RPA) reaction, and the templated reaction is a second RPA reaction. In other embodiments, the first RPA reaction is performed by incubating the RPA reaction mixture for 2 to 5 minutes at a temperature between 35 ° C and 45 ° C. In some embodiments, there is at least 100 times more of a substantially monoclonal template nucleic acid molecule present on the templated solid support than there is on the pre-seeded solid support.

[0016] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits, and apparatus for performing the methods, wherein the methods are for determining the sequence of a template nucleic acid molecule and comprise: a) performing a preimplantation reaction by incubating a first recombinase-polymerase amplification (RPA) reaction mixture under preimplantation reaction conditions to produce one or more preimplanted solid supports, the first recombinase-polymerase amplification reaction mixture comprising a population of template nucleic acid molecules and a population of solid supports comprising the same population of first primers attached, wherein the preimplantation reaction conditions comprise incubating the RPA reaction mixture under isothermal conditions, and wherein each of the preimplanted solid supports has between 10 and 100,000 substantially monoclonal nucleic acid molecules attached thereto, and / or the preimplantation reaction conditions comprise incubating the RPA reaction mixture under isothermal conditions for 2 to 5 minutes, wherein the preimplanted solid supports comprise: (i.) a substantially monoclonal population of template nucleic acid molecules attached to the solid support via the first primer, and (ii.) an attached first primer that is attached to the preimplanted solid support; The invention relates to a method for preparing a template reaction mixture comprising a first primer and a second RPA reaction mixture, wherein the template nucleic acid molecule is not associated with the pre-implanted solid carrier not contained in the template reaction mixture, wherein the template reaction mixture further comprises a second primer group in the form of a solution that is not connected to any substrate, and wherein the template nucleic acid molecule comprises a primer binding site of the second primer at or near the end opposite to the proximal segment comprising the first primer; c) starting the template reaction by adding a cation to the template reaction mixture; d) incubating the started template reaction mixture under isothermal conditions for at least 10 minutes to amplify the template nucleic acid molecule in the template reaction to produce one or more template solid carriers, wherein the one or more template solid carriers contain at least 10 times more substantially monoclonal template nucleic acid molecules on the template solid carrier than those present on the pre-implanted solid carrier; and e) sequencing the template nucleic acid molecules on the one or more template solid carriers to thereby determine the sequence of the template nucleic acid molecule. In some embodiments, the template nucleic acid molecule comprises two or more template nucleic acid molecules having different sequences. In some embodiments, the substantially monoclonal template nucleic acid molecules attached to the pre-implanted solid support and / or to the templated solid support comprise template nucleic acid molecules having two or more different sequences. In further embodiments, the substantially monoclonal template nucleic acid molecules attached to each pre-implanted solid support comprise at least 70% of all template nucleic acid molecules attached to each pre-implanted solid support. In some embodiments, the templated solid support is a templated bead, and the sequencing comprises distributing the bead in the wells of the solid support prior to performing the sequencing reaction.In some embodiments, each pre-implanted solid carrier of the solid carrier colony has between 10 and 50,000 substantially monoclonal template nucleic acid molecules connected thereto, and the one or more solid carriers are beads. In some embodiments, pre-implanted reaction mixture and / or templated reaction mixture further comprise recombinase auxiliaries. In other embodiments, recombinase auxiliaries are single-stranded binding proteins and / or recombinase load proteins. In some embodiments, at a temperature between 35 ° C and 45 ° C, incubate templated reaction mixture and / or pre-implanted reaction mixture. In some embodiments, incubate templated reaction mixture 10 to 60 minutes. In some embodiments, substantially monoclonal template nucleic acid molecules present on templated solid carriers are at least 100 times of substantially monoclonal template nucleic acid molecules present on pre-implanted solid carriers.

[0017] In some embodiments, the present disclosure generally relates to compositions, and systems, methods, kits and devices for compositions, wherein the composition comprises a templated reaction mixture comprising a pre-implanted solid carrier population, nucleotides, recombinases and polymerases, wherein the pre-implanted solid carrier population has between 10 and 50,000 substantially monoclonal template nucleic acid molecules comprising a first primer connected thereto, and further comprises a connected first primer, the connected first primer being connected to the pre-implanted solid carrier and not binding to the template nucleic acid molecules, wherein the reaction mixture does not comprise a cation capable of starting a recombinase-polymerase amplification reaction, and wherein at least 95% of the template nucleic acid molecules in the reaction mixture are connected to one or more solid carriers. In some embodiments, the template nucleic acid molecules comprise two or more template nucleic acid molecules having different sequences. In some embodiments, the substantially monoclonal template nucleic acid molecules connected to the pre-implanted solid carrier and / or connected to the templated solid carrier comprise template nucleic acid molecules having two or more different sequences. In some embodiments, the pre-implanted solid carrier is a pre-implanted bead. In some embodiments, the templated reaction mixture comprises a recombinase auxiliary protein. In other embodiments, the recombinase auxiliary protein is a single-stranded binding protein and / or a recombinase load protein. In some embodiments, a first recombinase-polymerase amplification (RPA) reaction is used to produce a pre-implanted solid support. In other embodiments, the first RPA reaction is performed by incubating the RPA reaction mixture for 2 to 5 minutes at a temperature between 35°C and 45°C. In some embodiments, the pre-implanted reaction mixture further comprises an identical second primer population in the form of a solution, and the template nucleic acid molecule comprises a primer binding site of the first primer at or near the first end. In some embodiments, the templated reaction mixture further comprises a cation capable of starting a recombinase-polymerase amplification reaction.

[0018] In some embodiments of the method for producing one or more templated carriers, such as solid carriers provided herein, at least some, most or all nucleic acids connected to the templated carrier or carrier produced in the method contain at least one modified nucleotide, and the at least one modified nucleotide includes a connector connected thereto, such as a first connector part. Such methods may further include connecting the templated carrier to magnetic beads, and the magnetic beads have a modified nucleotide of a nucleic acid connected to the templated carrier that can be combined, connected (link) or connected (attach) by a connector or connector of the modified nucleotide, a part (such as a binding partner or a second connector part) to form a bead assembly of a templated carrier and a magnetic bead. In some embodiments of these methods, by applying a magnetic field to the bead assembly, the bead assembly is separated from any element that does not contain magnetic beads, thereby separating the bead assembly from any such element and forming an enriched templated carrier population. In some embodiments of the method, the separated bead assembly is further placed under conditions of releasing the templated carrier from the magnetic beads, and analyzed in another method, such as a sequencing method.

[0019] Also provided herein is a method for preparing a device for analyzing nucleic acids, such as sequencing nucleic acids. In some embodiments, the method comprises: generating a nucleic acid containing a capture sequence portion, a template portion, and a primer portion containing a first linker portion; capturing nucleic acids on a carrier having a plurality of capture primers complementary to the capture sequence portion of the nucleic acid, such as a solid carrier such as a bead, for example, by hybridization; connecting the first linker portion of the captured nucleic acid to the second linker portion contained on the magnetic bead to form a bead assembly of the captured nucleic acid and the magnetic bead on the carrier; applying a magnetic field to the bead assembly, thereby separating the bead assembly from any element that does not contain magnetic beads; releasing the captured nucleic acid on the carrier from the magnetic beads; mixing the captured nucleic acid released on the carrier with the magnetic beads, the captured nucleic acid on the carrier not being attached (attach / link) or bound to the mixture; and incorporating the mixture into a device for analyzing the captured nucleic acid. In some embodiments, the mixture of the captured nucleic acid and the magnetic beads on the carrier is coated on a surface, such as a chip, such as a semiconductor chip, and a magnetic field is applied to the surface. The surface may contain micropores, and the captured nucleic acid on the support is loaded into the micropores by moving the magnetic beads on the surface when a magnetic field is applied to the surface. In some such embodiments, the size of the magnetic beads is such that the magnetic beads cannot enter the micropores.

[0020] In some embodiments of a method for preparing a device for analyzing nucleic acids, the method includes: generating a nucleic acid containing a capture sequence portion, a template portion, and a primer portion containing a first linker portion; capturing the nucleic acid on a support having a plurality of capture primers complementary to the capture sequence portion of the nucleic acid, such as a solid support, such as beads; connecting the captured nucleic acid to magnetic beads having a second linker portion to form a bead assembly such that the first and second linker portions are connected; and loading the bead assembly into a hole in the device using a magnetic field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1A to 1D is a graph showing the frequency distribution of read lengths in high-throughput sequencing after bulk isothermal amplification.

[0022] Figure 1A Shown are read lengths using Ion Sphere Particles (ISP) without pre-implanted templates (no template control (NTC)).

[0023] Figure 1B Shown are read lengths using an ISP pre-seeded with approximately 70 copies / ISP.

[0024] Figure 1C Shown are read lengths using an ISP pre-seeded with approximately 665 copies / ISP.

[0025] Figure 1D The read length using the ISP pre-seeded with about 4,170 copies / ISP is shown ( Figure 1D ).

[0026] Figures 2A-2F is a bar graph showing various metrics of high throughput sequencing following bulk isothermal amplification using the NTC P1 ISP and ISPs pre-implanted with about 70, about 665, and about 4,170 copies / ISP.

[0027] Figure 2A The ISP load percentages of NTC ISP and ISP pre-implanted with different template copy numbers are shown.

[0028] Figure 2B Shown are the percentages of usable reads from NTC ISP and ISP pre-seeded with different template copy numbers.

[0029] Figure 2C Shown are the numbers of total reads from NTC ISP and pre-seeded with different template copy numbers.

[0030] Figure 2DShown are the mean, median and mode of read lengths from NTC ISP and ISP pre-implanted with different template copy numbers.

[0031] Figure 2E Shown are the percentages of empty wells for NTC ISPs and ISPs pre-seeded with different template copy numbers.

[0032] Figure 2F The percentage of low-quality wells for NTC ISP and ISP pre-seeded with different template copy numbers is shown.

[0033] Figures 3A-3F is a bar graph showing various metrics of high throughput sequencing following pre-implantation amplification reactions using an ISP with no template control (NTC) and an ISP pre-implanted with about 82, about 775, and about 5,400 copies / ISP.

[0034] Figure 3A The ISP load percentages of NTC ISP and ISP pre-implanted with different template copy numbers are shown.

[0035] Figure 3B Shown are the percentages of usable reads from the NTC ISP and ISPs pre-seeded with different template copy numbers.

[0036] Figure 3C Shown are the numbers of total reads from NTC ISP and pre-seeded with different template copy numbers.

[0037] Figure 3D Shown are the mean, median and mode of read lengths from NTC ISP and ISP pre-implanted with different template copy numbers.

[0038] Figure 3E Shown are the percentages of empty wells for ISPs pre-seeded with different template copy numbers.

[0039] Figure 3F Shown are the percentages of low-quality wells for ISPs pre-seeded with different template copy numbers.

[0040] Figure 4 is a schematic diagram showing the primer extension reaction of the upper and lower strands of the template nucleic acid molecule.

[0041] Figure 5 , Figure 6 and Figure 7 An exemplary scheme for ligating a nucleic acid to a vector is shown.

[0042] Figure 8 is a diagram of an example method for preparing a sequencing device.

[0043] Fig. 9A diagram showing an example protocol for attaching nucleic acids to beads is shown.

[0044] Fig.10 is a graph of the results of sequencing runs performed on nucleic acid templates generated using several different methods.

[0045] Fig.11 is a diagram of an example method for directly capturing nucleic acids by binding beads to nucleic acids attached to linker moieties.

[0046] Fig.12 An exemplary scheme for ligating a nucleic acid to a vector is shown.

[0047] Fig.13 is a diagram of an example method for indirect capture of nucleic acids by binding beads to nucleic acids containing a single-stranded handle region.

[0048] The figures identified above are provided to be representative and non-limiting.

[0049] definition

[0050] As used in this disclosure, the following terms shall be understood to have the following meanings:

[0051] The term "monoclonal" and variants thereof, when used in reference to one or more polynucleotide populations, refers to a polynucleotide population in which about 50-99% or up to 100% of the members of the population share at least 80% identity at the nucleotide sequence level. As used herein, the phrase "substantially monoclonal" and variants thereof, when used in reference to one or more polynucleotide populations, refers to one or more polynucleotide populations in which the template polynucleotide molecule being amplified is the single most prevalent polynucleotide in the population. Thus, all members of a monoclonal or substantially monoclonal population are not necessarily identical or complementary to one another. For example, different portions of a polynucleotide template may be amplified or replicated to generate members of the resulting monoclonal population; similarly, a certain number of "errors" and / or incomplete extensions may occur during amplification of the initial template, thereby generating a monoclonal or substantially monoclonal population whose individual members may exhibit sequence variability in themselves. In some embodiments, low or non-substantial levels of mixing of non-homologous polynucleotides can occur during a nucleic acid amplification reaction of the present teachings, and thus a substantially monoclonal population can contain a minority of one or more polynucleotides (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.001% of a variety of polynucleotides). In certain instances, at least 90% of the polynucleotides in the population are at least 90% identical to the initial single template used as the basis for amplification to produce a substantially monoclonal population. In certain embodiments, the methods for amplification generate a polynucleotide population wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the members of the polynucleotide population share at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the template nucleic acid from which the population was generated. In certain embodiments, the methods for amplification generate a polynucleotide population wherein a sufficient majority of the polynucleotides share sufficient sequence identity to allow sequencing of at least a portion of the amplified template using a high throughput sequencing system.

[0052] In some embodiments, at least 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the members of the template nucleic acid molecules attached to the templated vector will share greater than 90%, 95%, 97%, 99% or 100% identity with the template nucleic acid molecule. In some embodiments, members of the nucleic acid population generated using any of the amplification methods hybridize to each other under high stringency hybridization conditions.

[0053] In some embodiments, the amplification method produces a substantially monoclonal population of nucleic acid molecules that contains sufficiently few polyclonal impurities so that it is successfully sequenced in a high-throughput sequencing method. For example, the amplification method can produce a substantially monoclonal population of nucleic acid molecules that produces a signal (e.g., a sequencing signal, a nucleotide incorporation signal, and the like) detected using a specific sequencing system. Optionally, the signal can then be analyzed to correctly determine the sequence and / or base identity of any one or more nucleotides present in any nucleic acid molecule of the population. Examples of suitable sequencing systems for detecting and / or analyzing such signals include the Ion Torrent sequencing system, such as the Ion Torrent PGM TM Sequence systems, including 314, 316, and 318 systems; Ion Torrent Proton TM Sequencing systems, including Proton I (Thermo Fisher Scientific, Waltham, MA); and Ion Torrent Proton TM Sequencing systems, including the Ion S5 and S5XL (Thermo Fisher Scientific, Waltham, Mass.) In some embodiments, the monoclonal amplicon permits accurate sequencing of at least 5 contiguous nucleotide residues on the Ion Torrent Sequencing System.

[0054] As used herein, the term "clonal expansion" and variants thereof refer to any method whereby a substantially monoclonal polynucleotide population is generated by amplifying a polynucleotide template. In some embodiments of clonal amplification, two or more polynucleotide templates are amplified to generate at least two substantially monoclonal polynucleotide populations.

[0055] As used herein, the term "preimplantation", also referred to herein as "implantation", refers to a process involving connecting one or more nucleic acid molecules to a carrier. In some embodiments, preimplantation involves connecting one or more nucleic acids to a carrier so that the connected nucleic acids can be further manipulated and / or analyzed, such as subjected to nucleic acid amplification, sequencing, and / or other processes. In some embodiments, the preimplantation process produces a single carrier, wherein a single nucleic acid molecule is connected thereto. A single carrier with a single nucleic acid molecule connected thereto may be included in a population of two or more carriers, a plurality of carriers, or a collection of two or more carriers, wherein each carrier has a single nucleic acid molecule connected thereto. In some embodiments, each such carrier has a different single nucleic acid molecule connected thereto. In some embodiments, during the preimplantation process, multiple copies of nucleic acid molecules are connected to the carrier and / or multiple different nucleic acids are connected to the carrier. In some embodiments, during the preimplantation process, a limited number of copies of substantially identical nucleic acids are connected to the carrier to produce a substantially monoclonal nucleic acid population. In some embodiments, the preimplantation of the carrier comprises the connection of the nucleic acid to the carrier, for example, by hybridization of the nucleic acid with a complementary polynucleotide connected to the carrier, without involving nucleic acid amplification. In some embodiments, the preimplantation of the carrier comprises nucleic acid amplification, for example, one or more cycles of nucleic acid amplification (e.g., PCR) and / or isothermal amplification. For example, nucleic acid amplification can be used in the pre-implantation process to produce one or more copies of the nucleic acid that can be connected (for example, by hybridization) to the carrier.Usually, the pre-implantation of the carrier that is connected with more than one nucleic acid template or multiple copies of nucleic acid template comprises nucleic acid amplification.The carrier produced in the pre-implantation or implantation process as provided herein is referred to as "pre-implanted" or "implanted" carrier.

[0056] As used herein, "limited number" generally refers to the number of different nucleic acids that are controlled for various purposes when referring to the number of nucleic acids (or copies of nucleic acids) that are attached to a carrier in a pre-implantation and / or templated method. A limited number of copies of a nucleic acid can be, for example, sufficient to provide a crowding effect in any subsequent larger-scale amplification (e.g., templated) of nucleic acids on a carrier to produce a larger substantially monoclonal nucleic acid population, so as to prevent or reduce the formation of a polyclonal population by preventing or reducing the migration of templates between reaction sites. Such limited number of template copies is limited so that a relatively short nucleic acid amplification time is used, for example, to prevent or reduce the migration of templates between reaction sites, but to produce a sufficient number of template copies to provide a crowding effect in subsequent amplification.

[0057] As used herein, the terms "comprises / comprising", "includes / including", "has / having" or any other variations thereof are intended to cover a non-exclusive inclusion. For example, a process, method, article or apparatus that includes a list of features is not necessarily limited to only those features but may include other features not expressly listed or inherent to such process, method, article or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not an exclusive or. For example, condition A or B is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0058] In addition, "a" or "an" is used to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. Unless otherwise clearly intended, this description should be understood to include one or at least one, and the singular also includes the plural.

[0059] Other features and advantages of the disclosure will be apparent from the following detailed description, and from the claims. DETAILED DESCRIPTION

[0060] In some embodiments, the disclosure generally relates to methods for manipulating nucleic acids and systems, compositions, kits and equipment for carrying out the methods. In some embodiments, the methods include nucleotide polymerization, nucleotide sequence modification (e.g., joining adapters and / or primer sequences, such as cleavable primers), nucleotide modification (e.g., adding labels or linker molecules (e.g., biotin) to one or more nucleotides in a nucleic acid molecule), nucleic acid amplification, nucleic acid restriction, nucleic acid capture, nucleic acid containment and / or nucleic acid transfer. The methods and compositions provided herein can be used, for example, to produce specific specific nucleic acid molecules, which are used, for example, to produce template nucleic acid populations that can be used, for example, for nucleic acid sequencing methods. In some examples, the methods and compositions provided herein can be used to produce monoclonal or substantially monoclonal nucleic acid populations. In some embodiments, the methods are used to produce monoclonal or substantially monoclonal nucleic acid populations, wherein nucleic acids are connected to carriers, such as solid carriers. In some embodiments, the methods are used to connect nucleic acids to two or more carriers. In such embodiments, two or more carriers can be the same or different, including, for example, polymerization and / or magnetic carriers. In some embodiments, the methods are used to transfer nucleic acids connected to one or more carriers to reaction sites and / or reaction chambers. In certain embodiments, the method is used to produce monoclonal or substantially monoclonal nucleic acid colonies in reaction sites and / or reaction chambers or multiple reaction sites and / or multiple reaction chambers. The method provided herein can be carried out alone or in any combination for any one or more purposes, and the advantages of nucleic acid analysis are provided. For example, the use of method and / or composition disclosed herein provides nucleic acid analysis results that improve quality, quantity and / or accuracy, such as nucleic acid sequencing results. In another example, method and / or composition disclosed herein are used alone and / or in any combination to enhance nucleic acid manipulation methods, to significantly improve workflow efficiency and / or promote nucleic acid analysis, such as the workflow automation of nucleic acid sequencing.

[0061] In some embodiments, the present disclosure generally relates to methods, systems, compositions, kits and devices for improving high-throughput nucleic acid sequencing results. The methods, systems, compositions, kits and devices are incorporated into methods and compositions for producing, containing, separating, transferring, replicating and / or manipulating substantially monoclonal nucleic acid populations, and the methods and compositions are fast, effective and cost-effective while providing, compared to existing methods, significantly increasing the production of high-quality nucleic acid sequencing reads and / or longer lengths of nucleic acid sequencing reads and reducing the number and running time of repetitive, non-information, errors and / or library reads. In some embodiments, the methods, systems, compositions, kits and devices include carriers, such as solid carriers, to constrain, enrich, mask, separate, locate, amplify and / or transfer nucleic acids that can be used in analytical methods. In some embodiments, the carrier is pre-implanted with a nucleic acid molecule or a limited number of mainly substantially identical nucleic acid molecules from a larger nucleic acid collection (e.g., a nucleic acid library or sample) to provide a separate carrier connected to a single nucleic acid molecule or to a local substantially monoclonal nucleic acid population. Such pre-implanted carriers are easy to manipulate and used as, for example, a single nucleic acid molecule or two or more substantially identical nucleic acid molecules, clean, closed, contained and / or separated sources, which can be cloned and amplified, for example, in a templated reaction, a relatively pure, closed, contained and / or separated nucleic acid template set is produced for a high-throughput sequencing workflow to improve sequencing results. In certain embodiments, pre-implanted carriers can be produced at least in part, and / or isothermal amplification, especially recombinase-mediated amplification reactions, such as recombinase-polymerase amplification (RPA) are used to amplify. Therefore, in some embodiments or aspects of the method for manipulating nucleic acids provided herein, for example, in a high-throughput sequencing workflow, a pre-implanted reaction is carried out before a templated reaction.

[0062] In a typical high-throughput sequencing workflow, nucleic acid molecules in a sample are used to prepare a template nucleic acid molecule library suitable for downstream sequencing. Multiple amplification can be optionally performed on nucleic acid molecules before, during, or after library preparation. Subsequently, the template nucleic acid molecule library is amplified onto one or more carriers for sequencing reactions. In some embodiments of the methods provided herein, amplification of a template on a carrier, such as a solid carrier, is typically performed in two or more reactions, including, for example, one or more pre-implantation reactions, wherein the one or more pre-implantation reactions produce one or more pre-implanted carriers having a template nucleic acid molecule connected thereto or a substantially monoclonal template nucleic acid molecule population connected thereto; subsequently, one or more templated reactions are performed on the pre-implanted carriers, wherein the one or more templated reactions produce more copies (e.g., at least 10× copies) of one or more connected template nucleic acid molecules on one or more carriers. The advantage of performing pre-implantation reactions and templated reactions is that this workflow produces higher quality sequencing reads in high-throughput sequencing reactions. In some embodiments, a pre-implantation reaction is performed with a capped primer to prevent the formation of primer dimer amplicons during the pre-implantation reaction. In previous methods, primer dimer amplicons can be generated during templating, which can produce lower quality sequencing reads and reduce the number of sequencing reads from the template nucleic acid molecule. Therefore, adding a separate pre-implantation reaction provides this and a number of other advantages over previous methods.

[0063] In some embodiments, the present disclosure generally relates to methods, systems, compositions, kits and equipment for amplifying template nucleic acid molecules, the methods, systems, compositions, kits and equipment comprising a templated reaction mixture comprising one or more pre-implanted vectors. In various aspects, the templated vectors provided herein are used to carry out downstream sequencing methods. The templated reaction typically uses a recombinase to denature or separate the double-stranded template nucleic acid molecules, and is carried out at an isothermal temperature with a polymerase suitable for RPA reactions. In some aspects, pre-implanted vectors are produced in separate pre-implanted reaction mixtures.

[0064] In certain embodiments, the pre-implantation (or implantation) method provided herein comprises a single-stranded nucleic acid molecule and a complementary nucleic acid, such as oligonucleotide hybridization, combined with a carrier and fixed on the carrier. Such methods are usually carried out in a short period of time under annealing conditions. In certain embodiments, the implantation method relates to a carrier such as a solid carrier, such as a bead or a particle, under which hybridization is connected with only a single-stranded nucleic acid molecule. Such conditions include, for example, contacting a single-stranded nucleic acid (for example, from a nucleic acid library or a sample) colony with a large amount of excessive carriers relative to the number of nucleic acid molecules under annealing conditions. For example, the ratio of carrier to template nucleic acid molecule is selected in some embodiments to optimize the percentage of the carrier of a substantially monoclonal template nucleic acid molecule colony with a single template polynucleotide molecule or connected thereto. For example, pre-implantation can be performed with a vector: template nucleic acid molecule ratio of at least about 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, or 100:1.

[0065] In some embodiments, the pre-implantation (or implantation) method provided herein comprises connecting the nucleic acid to the carrier by hybridization and amplifying the connected nucleic acid to a low level, such as a combined process of about 20 copies at the same time. In these embodiments, the pre-implantation reaction mixture generally comprises a population of template nucleic acid molecules, a polymerase, nucleotides, a first primer population, and a cofactor such as a divalent cation. It will be appreciated by those skilled in the art that various methods can be used to pre-implant a solid carrier with a substantially monoclonal template nucleic acid molecule. As a non-limiting example, RPA reactions, template walking reactions, PCR, emulsion PCR, or bridge PCR can be used to carry out pre-implantation reactions. Pre-implantation reactions and / or templated reactions can be carried out in large quantities in a solution. In addition, pre-implantation reaction mixtures and / or templated reaction mixtures may include: a first universal primer connected to one or more carriers; a second universal primer (soluble second universal primer) in the form of a solution; and a plurality of template nucleic acid molecules, wherein individual template nucleic acid molecules are joined to at least one universal primer binding sequence that can be added during library preparation, and wherein the universal primer binding sequence is combined with the first and optional second universal primers. In some embodiments, pre-implantation reactions and / or templated reactions are carried out in holes. In some embodiments, the pre-seeding reaction and the templating reaction are performed using sequential RPA reactions, wherein the template nucleic acid molecules are washed away after the pre-seeding reaction and before the templating reaction.

[0066] The carrier with nucleic acid molecules, such as beads, is modified to be loaded into a constrained area or container (receptacle), such as a micropore or a pit, to form an array presenting several nucleic acid templates and / or sequencing advantages. Placing the beads coated with nucleic acids in an organized, tightly packed manner, such as being placed in a small micropore, can increase the flux per cycle and reduce customer costs. As the density of the micropore increases or as the micropore size decreases, the bead loading becomes difficult, thereby producing many empty micropores and reducing the bead count in the hole. Too many empty micropores reduce the number of base reads and therefore poor sequencing performance. In certain embodiments, there is provided herein a method for introducing a carrier, such as a solid carrier, such as a bead, into a micropore or a reaction chamber, and a system, composition, kit and equipment for use in the method. In certain embodiments, the method comprises connecting a bead carrier having a modified captured template nucleic acid with a linker portion to a magnetic bead with a complementary linker portion to form a bead assembly, and using a magnetic field to load the bead assembly into the hole. The bead assembly can be denatured to release the magnetic beads so that the bead carrier is connected to the target nucleic acid in the hole. Such methods can be used, for example, in a device for preparing nucleic acid sequencing. In one embodiment, the reaction carried out in the hole can be an analytical reaction for identifying or determining the characteristics or properties of the analyte of interest. Such reactions can directly or indirectly produce a byproduct that provides a signal that can indicate whether the analyte reacts in a characteristic manner. If such byproducts are produced in small quantities or decay rapidly or react with other components, multiple copies of the same analyte can be analyzed in the hole at the same time to increase the output signal produced. In the embodiment of the method provided herein, before or after being placed in the hole, analyte, such as multiple copies of nucleic acid, can be connected to a solid phase carrier. For example, target nucleic acid can be amplified in a micropore to provide a clonal population of a target nucleic acid suitable for target nucleic acid sequencing. The solid phase carrier can be, for example, micron particles, nanoparticles, beads, solid or porous including gel or the like. For simplicity and ease of explanation, solid phase carriers are also referred to as particles or beads in this article. For nucleic acid analytes, multiple ligated copies can be prepared by rolling circle amplification (RCA), exponential RCA, or similar techniques to generate amplicons without the need for a solid support.

[0067] In some embodiments, the present disclosure generally relates to methods, systems, compositions, kits and devices for producing one or more templated solid carriers, the methods comprising: a) forming a templated reaction mixture by combining one or more pre-implanted solid carriers, nucleotides, recombinases and polymerases, wherein the one or more pre-implanted solid carriers comprise a substantially identical first primer population connected and have a template nucleic acid molecule connected thereto or have a substantially monoclonal template nucleic acid molecule connected thereto, wherein the template nucleic acid molecule comprises a proximal segment including the first primer, wherein the proximal segment connects the template nucleic acid segment to the solid carrier at the first primer, and wherein the pre-implanted solid carrier further comprises a connected first primer, the connected first primer is connected to the pre-implanted solid carrier and is not bound to the template nucleic acid molecule, wherein the templated reaction mixture further comprises a substantially identical soluble second primer population, and wherein the template nucleic acid molecule comprises a primer binding site of the second primer at or near the end opposite to the proximal segment; and b) performing one or more templated reactions. In some embodiments, the templated reaction is performed under isothermal conditions. In some embodiments, the templated reaction is performed by adding cations to the templated reaction mixture and incubating the reaction mixture under isothermal conditions. In some embodiments, the templated reaction mixture is incubated under isothermal conditions for at least 10 minutes to amplify the template nucleic acid molecules in the templated reaction to produce one or more templated solid supports. In some embodiments, the method provides templated solid supports, wherein each support comprises at least 100,000 substantially monoclonal template nucleic acid molecules.

[0068] In certain embodiments, two or more reactions are carried out in a templated method. In certain embodiments, two or more independent reactions are carried out in a templated method. For example, in some methods provided herein, a first or initial templated reaction is carried out, and then a second or subsequent templated reaction is carried out. In some cases, the initial templated reaction comprises one or more carriers, and one or more template polynucleotides are connected to the carrier, and the template polynucleotides are produced in a separate pre-implantation process before the templated reaction starts. The initial templated reaction comprises, for example, using a recombinase and a polymerase (i.e., RPA) to amplify one or more template polynucleotides on a pre-implanted carrier under substantially isothermal conditions. Compared with the duration of the subsequent second templated reaction, the initial first templated reaction is subsequently carried out, and the second or subsequent templated reaction is generally carried out in a shorter time period. For example, an initial first templated reaction followed by a second or subsequent templated reaction can be carried out for about 1-10 minutes, about 1-9 minutes, about 1-8 minutes, about 1-7 minutes, about 1-6 minutes, about 1-5 minutes, about 1-4 minutes, about 1-3 minutes, about 1-2.5 minutes, about 1-2 minutes, about 1-1.5 minutes, about 2-10 minutes, about 2-9 minutes, about 2-8 minutes, about 2-7 minutes, about 2-6 minutes, about 2-5 minutes, about 2-4 minutes, about 2-3 minutes, about 2-2.5 minutes, about 2.5-10 minutes, about 2.5-9 minutes, about 2.5-8 minutes, about 2.5-7 minutes, about 2.5-6 minutes, about 2.5-5 minutes, about 2.5-4 minutes or about 2.5-3 minutes. In some examples, the initial first templated reaction, followed by a second or subsequent templated reaction, can be carried out for less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2.5 minutes, less than about 2 minutes, less than about 1.5 minutes, or less than about 1 minute. In one non-limiting example, an initial templated reaction of about 2.5 minutes is carried out at about 40° C. using RPA. In some embodiments, the initial or first templated reaction is terminated or limited before starting the second or subsequent templated reaction. For example, a termination composition, such as a templated reaction inhibitor, can be added to an initial or first templated reaction that inhibits or stops the reaction and / or prevents the reaction from continuing. Examples of templated reaction inhibitors include, but are not limited to, one or more components that inhibit a nucleic acid amplification reaction, such as a polymerase inhibitor or a recombinase inhibitor, and / or a composition reaction of components required to limit the reaction. For example, in an initial templated reaction involving recombinase-polymerase nucleic acid amplification, the templated reaction inhibitor can be a chelator, such as a metal or cation chelator, such as EDTA, which binds a required cation, such as magnesium, for the recombinase-mediated reaction in the recombinase-polymerase amplification.In another example, the templated reaction can be limited by removing the reaction components, for example, by washing the templated reaction site, such as a reaction chamber or a hole, with a solution that does not contain one or more components required for the templated reaction. For example, the templated reaction site can be washed or rinsed with a solution that does not contain recombinase, polymerase, cation, nucleotide and / or other components of the RPA reaction for the templated reaction. In some embodiments, the second or subsequent templated reaction after the initial or first templated reaction is started or promoted by adding or contacting one or more templated reaction components to a template-bound carrier that has already performed an initial or first templated reaction. For example, in a second or subsequent templated reaction comprising a recombinase-polymerase nucleic acid amplification, one or more of the following components can be added to a template-bound carrier or contacted with a template-bound carrier: a recombinase, a polymerase, a nucleotide and / or a cation. The second or subsequent templated reaction after the first or initial templated reaction is generally carried out for a longer period of time than the duration of the first templated reaction. For example, a second or subsequent templating reaction following a first templating reaction can be carried out for about 5-60 minutes, about 5-50 minutes, about 5-45 minutes, about 5-40 minutes, about 5-35 minutes, about 5-30 minutes, about 5-25 minutes, about 5-20 minutes, about 5-15 minutes, about 5-10 minutes, about 10-60 minutes, about 10-50 minutes, about 10-45 minutes, about 10-40 minutes, about 10-35 minutes, about 10-30 minutes, In some examples, the second or subsequent templated reaction after the first or initial templated reaction can be carried out for at least about 60 minutes, at least about 55 minutes, at least about 50 minutes, at least about 45 minutes, at least about 40 minutes, at least about 35 minutes, at least about 30 minutes, at least about 25 minutes, at least about 20 minutes, less than 2 minutes, less than 1.5 minutes or less than 1 minute. In one non-limiting example, a second or subsequent templated reaction is performed using RPA at about 40° C. for about 20 minutes. In some embodiments, the second or subsequent templated reaction is terminated or limited at a set time. For example, a termination composition, such as a templated reaction inhibitor, can be added to a second or subsequent templated reaction that inhibits or stops the reaction and / or prevents the reaction from continuing. In another example, the templated reaction can be limited by removing reaction components, such as by washing the templated reaction site, such as a reaction chamber or a well, with a solution that does not contain one or more components required for the templated reaction.

[0069] An advantage of comprising two or more reactions to produce a substantially monoclonal nucleic acid population in the templated process is that it promotes the control of template amplification, which can reduce or prevent polyclonality in nucleic acid populations. For example, the initial or first amplification of the template polynucleotide on the pre-implanted carrier with limited quantity and / or duration can limit the amount of free templates that can be used to move or migrate to another templated reaction site. The initial or first templated reaction provides the combination of the template replicated with the other fixed primers on the pre-implanted carrier, and therefore provides a less open environment for the diffusion of template nucleic acid in the second or subsequent templated reaction amplification. In some embodiments in which two or more reactions are carried out in the templated method, one or more reactions in the reaction include one or more diffusion-restricted agents, and the example of the diffusion-restricted agent is provided in this article. The diffusion-restricted agent can further prevent or slow down the diffusion of the amplified polynucleotides produced by the replication of at least some parts of the template nucleic acid molecule in the templated reaction mixture, thereby preventing or reducing the nucleic acid in the templated reaction mixture for producing a monoclonal template population from being transferred to another templated reaction mixture for producing different monoclonal template populations, thereby reducing the formation of polyclonal contaminants during nucleic acid amplification in the templated reaction. In some embodiments, the diffusion-restricted agent included in one or more templated reactions is a sieving agent, such as a polymer, such as methylcellulose. In some embodiments, the diffusion-restricted agent included in one or more templated reactions is a pharmaceutical composition. For example, a drug compound can be a compound that binds or is connected to a template nucleic acid during a templated reaction, such as during a template nucleic acid amplification process, to reduce the mobility of the template nucleic acid in a templated reaction mixture. In some embodiments, the template nucleic acid is modified to include a portion (referred to as a "drug tag") that is bound to the drug compound. In a non-limiting example, the template nucleic acid is biotinylated and the drug compound included in one or more templated reactions is streptavidin or a derivative thereof, such as neutravidin. In some embodiments, the drug compound is included in a first or initial templated reaction. In some embodiments, the drug compound is included in a first or initial templated reaction, but is not included in a second or subsequent templated reaction.

[0070] In some embodiments, the template nucleic acid molecule is not included in the templated reaction mixture in the form of a solution. In certain embodiments, when starting the templated reaction, the template nucleic acid molecule is usually present in the reaction mixture in the form of a solution. Usually, a pre-implanted solid carrier is produced in the pre-implanted reaction separated from the templated reaction. In certain embodiments, the template nucleic acid is connected to the solid carrier by not comprising the proximal section of 100 or more identical nucleotides. In certain embodiments, the proximal section does not comprise a continuous sequence greater than 2,3,4,5,6,7,8,9 or 10 identical nucleotides. In some embodiments of the above aspects, one or more templated solid carriers are used in the sequencing reaction to determine the sequence of the template nucleic acid molecule.

[0071] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits and apparatus for determining the sequence of a template nucleic acid molecule, the methods comprising: a) generating a population of pre-implanted solid carriers comprising a population of substantially identical first primers attached, wherein the pre-implanted solid carriers are generated under pre-implantation conditions, and wherein each of the pre-implanted solid carriers has one template nucleic acid molecule or, for example, between 10 and 100,000 substantially monoclonal template nucleic acid molecules comprising a first primer attached thereto, and further comprising an attached first primer that is attached to the pre-implanted solid carrier and does not bind to the template nucleic acid molecule; b) attaching the pre-implanted solid carriers, the nucleic acid carriers to the pre-implanted solid carriers, and the pre-implanted solid carriers to the pre-implanted solid carriers; and c) attaching the pre-implanted solid carriers, the nucleic acid carriers to the pre-implanted solid carriers, and the pre-implanted solid carriers to the pre-implanted solid carriers. The invention relates to a method for preparing a templated reaction mixture comprising: combining a group of nucleotides, a recombinase and a polymerase to form a templated reaction mixture; c) starting a templated reaction by adding a cation to the templated reaction mixture, wherein the template nucleic acid molecule is not present in the reaction mixture in solution when the templated reaction is started; d) incubating the started templated reaction mixture under isothermal conditions for at least 10 minutes to amplify the template molecule in the templated reaction to produce one or more templated solid carriers, wherein the one or more templated solid carriers contain at least 10 times the number of attached substantially monoclonal template nucleic acid molecules on the templated solid carriers as those present on the pre-implanted solid carriers; and e) sequencing the template nucleic acid molecules on the one or more templated solid carriers to thereby determine the sequence of the template nucleic acid molecules.

[0072] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits, and apparatus for determining the sequence of a template nucleic acid molecule, the methods comprising: a) generating one or more pre-implanted vectors comprising linked substantially identical first primers, wherein the pre-implanted vectors are generated under pre-implanted conditions, and wherein each pre-implanted vector has one template nucleic acid molecule or more (e.g., between 10 and 100,000) substantially monoclonal template nucleic acid molecules comprising a first primer linked thereto, and further comprising a linked first primer that is linked to the pre-implanted vector and does not bind to the template nucleic acid molecule; b) forming a first templated reaction mixture by combining the pre-implanted vectors, nucleotides, a recombinase, and a polymerase; c) initiating a first templated reaction; d) incubating the initiated first templated reaction mixture under isothermal conditions to amplify the template molecules in the templated reaction to generate one or more templated vectors having more linked substantially monoclonal template nucleic acid molecules thereon than present on the pre-implanted vectors; and e) sequencing the template nucleic acid molecules on the one or more templated vectors, thereby determining the sequence of the template nucleic acid molecules. In some embodiments of such methods for determining the sequence of a template nucleic acid molecule, when the template reaction is initiated, the template nucleic acid molecule is not present in solution in the reaction mixture. In some embodiments, such methods, and systems, compositions, kits, and apparatus for determining the sequence of a template nucleic acid molecule further comprise after step d) and before step e): (i) stopping, interrupting, or limiting the first template reaction, (ii) forming a second template reaction mixture by combining the template vector, nucleotides, recombinase, and polymerase from the first template reaction; (iii) starting or promoting the second template reaction; (iv) incubating the started second template reaction mixture under isothermal conditions to amplify the template molecule in the template reaction to produce one or more template vectors having more attached substantially monoclonal template nucleic acid molecules than present on the template vector from the first template reaction. In some embodiments, one or more or all template reactions are performed in a reaction chamber, such as a well or microwell.

[0073] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits, and apparatus for determining the sequence of a template nucleic acid molecule, the methods comprising: a) performing a preimplantation reaction by incubating a recombinase-polymerase amplification (RPA) reaction mixture comprising a population of template nucleic acid molecules and a population of solid supports comprising a population of substantially identical first primers attached under preimplantation reaction conditions to produce one or more preimplanted solid supports, the one or more preimplanted solid supports comprising a substantially monoclonal population of template nucleic acid molecules attached to the solid support via a first primer and an attached first primer, the attached first primer being attached to the preimplanted solid support and not binding to the template nucleic acid molecule, wherein the preimplantation reaction conditions comprise incubating the RPA reaction mixture under isothermal conditions, wherein each of the preimplanted solid supports has between 10 and 100,000 substantially identical first primers attached thereto. monoclonal nucleic acid molecules, and / or the pre-implantation reaction conditions include incubating the RPA reaction mixture under isothermal conditions for 2 to 5 minutes; b) forming a templated reaction mixture by including one or more pre-implanted solid carriers in the RPA reaction mixture, wherein template nucleic acid molecules not associated with the pre-implanted solid carriers are not included in the templated reaction mixture; c) starting the templated reaction by adding cations to the templated reaction mixture; d) incubating the initiated templated reaction mixture under isothermal conditions for at least 10 minutes to amplify the template nucleic acid molecules in the templated reaction to produce one or more templated solid carriers, wherein the one or more templated solid carriers contain at least 10 times more substantially monoclonal template nucleic acid molecules on the templated solid carriers than those present on the pre-implanted solid carriers; and e) sequencing the template nucleic acid molecules on the one or more templated solid carriers to thereby determine the sequence of the template nucleic acid molecules.

[0074] In some embodiments, the multiple template nucleic acid molecules include two or more template nucleic acid molecules with different sequences. In some embodiments, the substantially monoclonal template nucleic acid molecules connected to the pre-implanted solid carrier and / or connected to the templated solid carrier include template nucleic acid molecules with two or more different sequences. Substantially monoclonal template nucleic acid molecules are usually connected to the solid carrier by primers, and the primers may include continuous identical nucleotides, or no continuous identical nucleotides or no more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 continuous nucleotides; or connected to the solid carrier by primers comprising continuous non-identical nucleotides. However, in some embodiments, the proximal section of the template nucleic acid includes less than 100 identical nucleotides, and the proximal section is a section connected to the solid carrier and is generally a primer. In some embodiments, the templated solid carrier is a templated bead, and sequencing is included in the distribution of beads in the holes of the solid carrier before performing the sequencing reaction. In some embodiments, each pre-implanted solid carrier in the solid carrier population can have, for example, between 10 and 50,000 or between 100 and 25,000 substantially monoclonal template nucleic acid molecules connected thereto. In illustrative examples, pre-implantation reaction mixture and / or templated reaction mixture further include the same second primer population in solution form. In these examples, the template nucleic acid molecule is generally included in the first end or the first primer near the primer binding site and the other end or the second primer near the primer binding site.

[0075] In another aspect, the templated reaction mixture comprises a pre-implanted population of solid supports, nucleotides, a recombinase, and a polymerase, wherein the pre-implanted population of solid supports has one template nucleic acid molecule or between 10 and 50,000 substantially monoclonal template nucleic acid molecules per support, wherein the template nucleic acid molecule comprises a first primer attached thereto, and the pre-implanted solid supports further comprise an attached first primer that is not bound to a template nucleic acid molecule, wherein the reaction mixture does not comprise a cation capable of initiating a recombinase-polymerase amplification reaction, and wherein at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% of the template nucleic acid molecules in the reaction mixture are attached to one or more solid supports.

[0076] In some embodiments, a pre-implantation reaction mixture is used to form one or more pre-implanted carriers during the pre-implantation reaction. In some embodiments, the pre-implantation reaction mixture comprises some or all of the following: a template nucleic acid molecule population, one or more carriers, a polymerase, a first primer population, nucleotides, and a divalent cation. In some embodiments, the pre-implantation reaction mixture comprises one or more template nucleic acid molecules, one or more carriers, and a first primer population. In some embodiments, the first primer population is connected to one or more carriers. In some embodiments, the pre-implantation reaction mixture comprises one or more, multiple, or a population of template nucleic acid molecules, such as a single-stranded template nucleic acid, and one or more or more carriers with multiple first primers are connected thereto, wherein the template nucleic acid molecule contains a nucleotide sequence complementary to the first primer, and the pre-implantation reaction mixture optionally comprises a polymerase and nucleotides. In any embodiment of the present invention, the pre-implantation reaction mixture further comprises a second primer and optionally a diffusion-restricted agent. In an illustrative embodiment, the second primer is in a solution. In some embodiments, the pre-implantation reaction mixture comprises a recombinase and optionally a recombinase auxiliary protein.

[0077] In some embodiments, pre-implantation and / or templated reactions are performed using nucleic acid amplification using: (i) multiple nucleic acid molecules comprising a target sequence and one or more universal adapter sequences; (ii) multiple soluble forward primers; (iii) multiple soluble reverse-terminated tailed primers; and (iv) multiple solid supports having capture primers fixed thereon, which hybridize to the universal adapter sequences. In some embodiments, pre-implantation and / or templated reactions are performed in a single reaction mixture having multiple nucleic acid molecules containing the same or different target sequences. In some embodiments, individual nucleic acid molecules generated from a sample comprise a target sequence joined to at least one universal adapter sequence (e.g., A-adapter and / or P1-adapter sequence) at the end. In some embodiments, the nucleic acid molecules are double-stranded molecules having complementary upper and lower chains. In some embodiments, a pre-implantation reaction is performed using forward and reverse soluble primers that hybridize to the adapter sequences to amplify the nucleic acid molecules and connect substantially monoclonal copies of the nucleic acid molecules to a solid support (see, e.g., Figure 4 ).although Figure 4 A series of reactions of a single double-stranded nucleic acid molecule and a single bead within a single reaction mixture is depicted, but one skilled in the art will appreciate that the same single reaction mixture contains multiple double-stranded nucleic acid molecules and multiple beads subjected to the same series of reactions to produce at least two beads each attached to a substantially monoclonal population of target sequences. Additionally, one skilled in the art will appreciate that a bead can be attached to multiple B capture primers. Further, Figure 4The lower side of depicts a biotinylated primer extension product bound to the B capture primer, but it will be appreciated by those skilled in the art that a non-biotinylated primer extension product can bind to the B capture primer. It will also be appreciated by those skilled in the art that a mixture of biotinylated and non-biotinylated primer extension products can be linked to multiple B capture primers linked to beads. In some embodiments, a single reaction mixture contains multiple nucleic acid molecules, wherein individual nucleic acid molecules having the same or different target sequences are linked to at least one universal adapter sequence. Reference is now made to Figure 4 , denaturing the nucleic acid molecules having the upper and lower strands, and using the separated upper and lower strands in a primer extension reaction using a soluble primer (e.g., a soluble A primer) and a soluble capped tailed primer (e.g., a soluble capped tailed P1 / B primer) to generate a plurality of primer extension products having an adapter sequence that can bind to the immobilized B primer during the pre-implantation and / or templated reaction. Figure 4 As shown in , the primer extension reaction produces different products for the two strands due to the different sequences and orientations of the top and bottom strands and the differences in the primers used.

[0078] In some embodiments, in the first primer extension reaction, a soluble primer that binds to the A primer binding site is used to generate the complementary strand of the bottom strand. Figure 4 (left), the soluble A' primer is complementary to the A adaptor sequence. In some embodiments, a mixture of soluble A' primers of varying lengths is used to perform the first primer extension reaction. The mixture of soluble A' primers may vary in length at its 5' end, 3' end, or both 5' and 3' ends. For example, primer mixture S (with or without a 5' biotin adduct (at Figure 4 A mixture of A' primers (described as "Bio"), which may contain 5' non-complementary sequences of various lengths and can be used in primer extension reactions to produce one of several possible first extension products (depending on which soluble A' primer is used to perform the first primer extension reaction) Figure 4 For example, in the 5' to 3' direction, the first extension product contains a complementary A-adaptor sequence ( Figure 4 A' in the left side), the complementary sequence (in Figure 4 The bottom ') and the complementary P1 sequence (shown in the left Figure 4Shown as P1′ on the left). In some embodiments, in the second primer extension reaction, the newly synthesized P1′ sequence in the first extension product can bind to the soluble P1 primer to allow primer extension from the 3′ end of the P1′ sequence of the first primer extension product. The soluble P1 primer can be a tailed primer. The soluble P1 primer can carry a blocking portion at its 3′ end, wherein the blocking portion can inhibit primer extension from the 3′ end of the primer. The soluble P1 primer can be a reverse tailed P1 primer, which includes a 5′B adapter sequence attached, such that using the tailed primer P1 as a template, primer extension of the first extension product causes the addition of a complementary sequence of the B sequence to the 3′ end (at Figure 4 In an illustrative embodiment, the soluble P1 primer may have a 3' blocked end (at Figure 4 left, shown as a circled "X") to prevent extension from the 3' end of the soluble P1 primer ( Figure 4 The second primer extension reaction can generate a plurality of second extension products of various lengths, depending on which soluble A' primer is used in the first extension reaction. In the 5' to 3' direction, the plurality of second extension products contain complementary A-adapter sequences (in Figure 4 A' on the left), the complementary bottom strand sequence (in Figure 4 The left side is shown as the lower '), the complementary P1 sequence (in Figure 4 The left side shows P1') and the complementary B adapter sequence (in Figure 4 The second primer extension product may contain or lack a 5' biotin adduct ( Figure 4 Left side). The second extension reaction can produce multiple second extension products, which have different lengths and may contain or lack biotin adducts and contain B' adapter sequences. Any of these second extension products can bind / hybridize with the B capture sequence immobilized on the solid surface (beads). The immobilized B primer can undergo a third primer extension reaction, thereby producing a third extension product ( Figure 4 lower side).

[0079] Reference Figure 4 (right side), which depicts a series of reactions for denaturation and stranding of double-stranded nucleic acid. In some embodiments, the P1′ adapter sequence of the strand can bind to a soluble P1 primer and undergo a first primer extension reaction to produce a first extension product ( Figure 4 Right side). In some embodiments, the soluble P1 primer is a tailed primer. The soluble P1 primer may carry a blocking portion at its 3′ end, wherein the blocking portion may inhibit primer extension from the 3′ end of the primer ( Figure 4The soluble P1 primer may be a tailed P1 primer comprising a 5′B adapter sequence attached such that primer extension using the tailed primer P1 as a template results in the addition of a complementary sequence (B′) of the B sequence to the 3′ end of the first extension product ( Figure 4 In an illustrative embodiment, the soluble P1 primer may have a 3′ blocked end (at Figure 4 on the right, shown as a circled "X") to prevent extension from the 3' end of the P1 primer ( Figure 4 The first primer extension reaction generates a plurality of first extension products, which contain an A′ adapter sequence, an upper strand sequence, a P1′ adapter sequence, and a B′ adapter sequence in the 5′ to 3′ direction ( Figure 4 The first extension product comprising the B′ adapter sequence can bind / hybridize to the B capture sequence immobilized on the solid surface (bead). The immobilized B primer can undergo a second primer extension reaction, thereby generating a second extension product ( Figure 4 lower side).

[0080] In some embodiments, Figure 5The pre-implantation (or implantation) reaction is shown. In this example, in the presence of a bead carrier with a capture primer, a target polynucleotide B-A' and its complementary sequence template polynucleotide (A-B') are amplified. The target polynucleotide has a capture portion (B) that is identical or substantially similar to the sequence of the capture primer coupled to the bead carrier. A substantially similar sequence is a sequence whose complementary sequence can hybridize with each of substantially similar sequences. The bead carrier may have a capture primer with a sequence identical or substantially similar to the sequence of the B portion of the target polynucleotide to allow the complementary sequence of the capture portion (B) of the target polynucleotide to hybridize with the capture primer connected to the bead carrier. Optionally, the target polynucleotide may include a second primer position (P1) adjacent to the capture portion (B) of the target polynucleotide, and may further include a target region adjacent to the primer and bound to the sequencing primer portion (A) of the target polynucleotide through the complementary sequence portion (A'). When amplified in the presence of a bead carrier comprising a capture primer, a template polynucleotide complementary to the target polynucleotide can hybridize with the capture primer (B). The target polynucleotide may remain in the solution. The system may be extended, wherein the capture primer B complementary to the template polynucleotide is extended to obtain a target sequence bound to the bead carrier. In the presence of a carrier with a capture primer, one or more additional amplifications may be performed at this stage. One or more additional amplifications may be performed in the presence of a free primer (B), a bead carrier, and a free modified sequencing primer (A) having a linker portion (L) attached thereto. Primer (B) and modified primer (LA) may interfere with the free floating target polynucleotide and the template polynucleotide, preventing them from binding to the bead carrier and to each other. Specifically, the modified sequencing primer (A) having a linker portion attached thereto may hybridize with the complementary portion (A') of the target polynucleotide connected to the bead carrier. Optionally, the linker-modified sequencing primer LA hybridized to the target polynucleotide may be extended to form a linker-modified template polynucleotide. Such linker-modified template polynucleotides are hybridized with target nucleic acids attached to the bead carrier, which may then be captured by magnetic beads and used for magnetic loading of a sequencing device. Amplification or extension can be performed using polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), or other amplification techniques. In a specific example, PCR amplification is used to perform Figure 5 Each step of the scheme is illustrated in the figure.

[0081] In some embodiments, Figure 6The pre-implantation (or implantation) reaction is performed as shown in . In this example, the alternative comprises a target polynucleotide (P1-A') and its complementary sequence template polynucleotide (A-P1'). In a solution comprising a linker-modified sequencing primer (LA) and a truncated P1 primer (trP1) having a portion of a sequence containing a capture primer (B), the target polynucleotide and the template polynucleotide are amplified. In an example, the truncated P1 primer (trP1) comprises a subset of the P1 sequence or all sequences P1. Subsequently, during amplification in the presence of a linker-modified sequencing primer (LA) and a truncated P1 primer (trP1-B), a single species 702 comprises a linker-modified template polynucleotide (LA-B') operable to hybridize with a bead carrier having a capture primer (B). Therefore, the linker-modified template polynucleotide (LA-B') hybridizes with the capture primer (B) on the bead, and extends to form a target polynucleotide (B-A') connected to the bead carrier. The linker-modified template polynucleotides that hybridize to the target polynucleotides attached to the beads can be attached to the magnetic beads, which can be used, for example, to implement magnetic loading of the beads into a sequencing device and / or to enrich the nucleic acids attached to the beads. The linker portion of the linker-modified template polynucleotide can take various forms, such as biotin, which can be combined with the linker portion attached to the magnetic beads, such as streptavidin. Each of the amplification reactions can be performed using PCR, RPA or other amplification techniques. In Figure 6 In the example shown in , three polymerase chain reaction (PCR) cycles can be used to perform the embodiment. This series of PCR reactions produces a higher percentage of bead carriers with a single target polynucleotide attached thereto. Therefore, more monoclonal populations can be generated in the wells in the sequencing device.

[0082] In some embodiments, Figure 7 The pre-implantation (or implantation) method is shown in . In this example, the method is designed to generate the desired vector-linked nucleic acid molecules from a series of amplification cycles, wherein only one of the amplification products (which is the desired target nucleic acid) will be linked to the vector. The desired target contains a linker moiety, such as biotin (in Figure 7 in, marked with the letter "L"); and at the 3' end with a primer fixed to the support (in Figure 7 , marked with the letter "B") is complementary to the adaptor nucleotide sequence (in Figure 7 In contrast, Figure 6 The method shown produces two nucleic acid amplification products that hybridize to the carrier, only one of which has the desired linker portion. In terms of producing only one amplification product that will be connected to the carrier, Figure 7The method described in avoids the production of vectors that do not contain the desired target nucleic acid (e.g., nucleic acid lacking a linker portion that will not be used for downstream analysis). Thus, this method avoids wasting vectors and nucleic acids and ensures that only a single nucleic acid target molecule will hybridize to the vector, which is desirable for maintaining high levels of monoclonality and subsequent templated amplification using a vector with only one nucleic acid template bound thereto. Figure 7 As shown in , the double-stranded library nucleic acid contains a different adapter sequence at each end, shown as an A adapter sequence at the 5' end and a P1 adapter sequence at the 3' end (e.g., standard Ion Torrent A and P1 library adapters; Thermo Fisher Scientific). To begin the implantation process, the library nucleic acid is subjected to one cycle of amplification (i.e., denaturation, primer annealing, and primer extension) in the presence of primers, such as Figure 7 As shown. Exemplary primers for amplification are biotinylated primer A (forward primer) and reverse fusion primer. Fusion primer (e.g., Figure 7 The primer labeled as trP1 in the example is a fusion of a sequence complementary to a portion of the adapter sequence at the 3' end of the target nucleic acid and a B primer sequence identical to the sequence of the B primer fixed on the support. Figure 7 In the example shown, trP1 is a 23-mer fragment of the Ion P1 adapter having the sequence of SEQ ID NO: 1. The fusion primer will hybridize and prime at the internal portion of the 3' adapter sequence of the library nucleic acid molecule close to the library insert sequence, and will not hybridize to the rest of the adapter sequence at the extreme 3' end of the library nucleic acid. This forms a mismatched end between the fusion primer sequence and the extreme 3' end portion of the adapter on the library nucleic acid. Figure 7 As shown in , after two cycles of amplification (e.g., PCR), although four amplification products are produced, only one product will be able to implant (or hybridize) to the carrier (e.g., ionic spherical particles). Therefore, after subsequent denaturation of the amplification products, only a single strand of one of the products will hybridize to the B primer on the carrier. This primer can be extended to form a double-stranded template nucleic acid, one of which contains a linker portion that can be used, for example, to bind the carrier-bound nucleic acid to magnetic beads for enrichment and / or magnetic loading of the pores. Although Figure 7 A series of reactions of a single double-stranded nucleic acid molecule and a single vector within a single reaction mixture is depicted, but one skilled in the art will appreciate that the same single reaction mixture contains multiple double-stranded nucleic acid molecules and multiple vectors subjected to the same series of reactions to produce at least two vectors each linked to a target template nucleic acid.

[0083] In some embodiments, the pre-implantation (or implantation) method is substantially as follows Figure 5-7, wherein one or more amplification (e.g., PCR) cycles are added to the method. Thus, in any such pre-implantation method employing multiple amplification cycles (e.g., PCR), two or more, e.g., 3, 4, 5 or more amplification cycles may be included in the pre-implantation method. For example, in the event that the maximum possible nucleic acid library input is below the optimal range, additional amplification cycles may be included in the implantation process to produce a sufficient amount of template implantation vectors, which can be used for additional methods, including, for example, templated amplification to produce a substantially monoclonal nucleic acid template population and a downstream sequencing process. Such situations may include, for example, a lower than expected library concentration caused by the library preparation method. Although in this case, the library template copy number can be increased to a more optimized level by expanding the implantation reaction to accommodate a larger library input amount, this is sometimes not an available option due to volume limitations in the reaction vessel or processing. An increased number of amplification cycles may be included at any point in the pre-implantation method, such as before and / or after the vector having the oligonucleotide primer attached thereto is introduced into the amplification protocol. For example, reference Figure 7 In the method described in, if an additional amplification cycle is included prior to the point where the vector is introduced into the reaction mixture, a total of four amplification products will produce nucleic acid chains that will hybridize to the B primer on the vector, compared to only one product that will produce a single chain as in the scheme shown without adding another amplification cycle. In addition, each of the four product chains will contain a linker portion attached to the 5' end of the nucleic acid. Therefore, after the added amplification cycle, when the vector with the B primer fixed is added to the reaction mixture, the next amplification cycle of denaturation, primer hybridization and primer extension will result in all four amplification product chains containing a sequence complementary to the B primer on the vector that hybridizes to the vector and is extended, thereby implanting four vectors compared to one vector. If in Figure 7 After the last amplification cycle shown, an additional amplification cycle is included, in which the carrier connected with the B primer is added to the reaction mixture, then a total of four amplification products will produce a nucleic acid chain, hybridized with the B primer on the carrier, by contrast, only one product produces a chain, hybridized as shown in the scheme, and another amplification cycle is not added. In addition, each of the four product chains will include the linker portion connected to the 5' end of the nucleic acid. Therefore, after the added amplification cycle, assuming that a sufficient number of carriers with the B primer fixed thereto are added to the reaction mixture, a total of four carriers will be implanted with nucleic acid templates compared to an implanted carrier if not including additional amplification. Therefore, by increasing the number of amplification cycles in pre-implantation methods such as these, more number of implantation carriers are obtained from the same number of input library nucleic acid molecules.

[0084] In some embodiments, beads having moieties on their surfaces that bind or attach to linkers (e.g., biotin) on target template nucleic acids are used to directly capture and enrich the implanted target nucleic acids after pre-implantation (implantation) and / or templated reactions. For example, in some embodiments, beads are prepared using soluble blocked tailed P1 / B primers (e.g., Figure 4 ) or biotinylated primer A (e.g., as depicted in Figure 7 The target template nucleic acid carrying the biotin adduct produced in the implantation reaction (as depicted in ), hybridizes with the primer fixed on the support, and then extends to form a double-stranded target template molecule bound to the support. The implanted support is then contacted with streptavidin-coated beads, such as magnetic beads, which bind the target nucleic acid via the biotin adduct to form a bead assembly (see Fig.11 ), thereby capturing the implanted carrier. The bead assembly can then be separated from any other reaction components, for example, by granulating the bead assembly with a magnet. The streptavidin-coated beads are subsequently separated from the target template nucleic acid (for example, by denaturing the double-stranded target bound to the solid carrier) and the target template nucleic acid bound to the solid carrier is removed from the separated beads to produce an abundant collection of single-stranded template-bound solid carriers. Excessive streptavidin-coated magnetic beads can be included in the capture process to ensure that all template nucleic acid-implanted carriers are captured. Any implanted amplification reaction product that carries a linker (for example, biotin) but is not bound to the carrier can be captured by the magnetic beads and eluted therefrom together with the implanted carrier, in further downstream processing (for example, in loading the implanted carrier into a reaction site, such as a micropore on a surface, such as a chip, for sequencing the template bound to the solid carrier, as described herein.

[0085] In some embodiments, Fig.12 The pre-implantation (or implantation) method is shown in . In this example, the method is designed to produce the desired solid support-attached nucleic acid molecules from a series of amplification cycles of the target nucleic acid, wherein only one of the amplification products (which is the desired target nucleic acid) will be attached to the support. The desired target contains a single-stranded nucleotide sequence at the 5' end of the nucleic acid, which is called the "handle" portion, and at the 3' end, which is attached to the primer fixed to the support (in Fig.12 The adaptor nucleotide sequence complementary to the Fig.12 In the example Fig.12 As shown in , the double-stranded library target nucleic acid contains an adapter sequence at each end, such as an A adapter sequence at the 5' end and a P1 adapter sequence at the 3' end (standard Ion Torrent A and P1 library adapters; Thermo Fisher Scientific). In some cases, such as Fig.12As shown, the input library may already have a handle configuration on the A adaptor. If the input library does not have a handle configuration on the A adaptor, it can be added to the library amplification reaction, which includes primers that contain a handle nucleotide sequence, a non-replicable portion (e.g., a polymerase stop site), and an A adaptor sequence in the 5' to 3' direction. To start the pre-implantation amplification reaction, the library is subjected to one amplification cycle (i.e., denaturation, primer annealing, and primer extension) in the presence of primers, as shown. Fig.12 As shown. Exemplary primers for amplification are primer A (forward primer), which has a polymerase termination site at its 5' end and a handle sequence located 5' of the non-replicable portion, and a reverse fusion primer. The non-replicable portion of the primer containing the handle can be any composition that cannot be replicated by the polymerase. Such non-replicable portions include, for example, any portion that cannot support template-based nucleotide polymerization by a polymerase. For example, the non-replicable portion can include a non-nucleotide portion (e.g., PEG or other carbon-based spacers, amino acids, or nucleotide analogs that are not recognized by the polymerase for primer extension). When a primer containing a handle is used for template-dependent nucleic acid synthesis by a polymerase, the polymerase cannot extend the synthesized nucleic acid chain beyond the non-replicable portion. This typically results in the cessation or termination of nucleic acid synthesis, and the non-replicable portion acts as a polymerase termination site. Fusion primers (e.g., Fig.12 The primer labeled as trP1 in the above) is a fusion of a sequence complementary to a portion of the adaptor sequence at the 3' end of the target nucleic acid and a B primer identical to the primer immobilized on the solid support. Fig.12 In the example shown, trP1 is a 23-mer fragment of the Ion P1 adapter having the sequence of SEQ ID NO: 1. The fusion primer will hybridize and prime at the internal portion of the 3' adapter sequence of the library nucleic acid molecule close to the library insert sequence, and will not hybridize to the rest of the adapter sequence at the extreme 3' end of the library nucleic acid. This forms a mismatched end between the fusion primer sequence and the extreme 3' end portion of the adapter on the library nucleic acid. Fig.12 As shown in , after two cycles of amplification (e.g., PCR), although four amplification products are produced, only one product will be able to implant (or hybridize) to the carrier (e.g., ionic spherical particles). Therefore, after subsequent denaturation of the amplification products, only a single strand of one of the products will hybridize to the B primer on the carrier. This primer can be extended to form a double-stranded template nucleic acid, one of which contains a handle that can be used, for example, to bind the carrier-bound nucleic acid to magnetic beads for enrichment and / or magnetic loading of the pores.

[0086] exist Fig.12In some embodiments of the pre-implantation (or implantation) method depicted in , the primer containing the handle sequence comprises in the 3' to 5' direction a first nucleotide sequence complementary to the sequence of the first double-stranded adapter at one end of the library DNA amplicon, a polymerase termination site, and a second nucleotide sequence (handle sequence) that is at least partially complementary or completely complementary to the first nucleotide sequence. The handle sequence may, for example, be complementary to a portion of the first nucleotide sequence, but contain one or more mismatched bases within the portion that are not complementary to bases in corresponding positions of the portion of the first nucleotide sequence. Under certain permissive conditions, the first nucleotide sequence and the second nucleotide sequence will hybridize to each other to form a hairpin structure. However, under the conditions of the amplification reaction that occurs during the implantation process, for example under Fig.12 In the method described in, the first and second sequences of nucleotides of the primer containing the handle will not hybridize. In contrast, under such non-permissive conditions, hybridization of the first nucleotide sequence of the primer with the complementary adapter sequence (e.g., A adapter) of the library amplicon will be favorable and occur, and the handle sequence will extend from the duplex of the A adapter and the first nucleotide sequence of the primer at the site of the polymerase termination portion as a single-stranded portion that is not replicated in the implant amplification reaction. Permissive conditions under which the first nucleotide sequence and the second nucleotide sequence of the primer containing the handle can hybridize can include, for example, a relatively low temperature, such as a temperature below the temperature at which the implant amplification reaction is performed or below the standard PCR operating temperature. Low temperature includes a temperature that is substantially or significantly lower than the Tm of the duplex double-stranded nucleic acid of the A adapter and the first nucleotide sequence of the primer. In some embodiments, the Tm of the duplex nucleic acid of the first nucleotide sequence and the second nucleotide sequence of the primer containing the handle is substantially or significantly less than the Tm of the duplex nucleic acid of the A adapter and the first nucleotide sequence of the primer. For example, the Tm of the hybrid formed between the first nucleotide sequence of the primer containing the handle and the second nucleotide sequence can be less than about 50°C, 45°C, 40°C, 35°C, 30°C, 25°C, 20°C or lower. Another example of a permissive condition is a relatively high salt concentration, such as a NaCl concentration of 0.25M, 0.3M, 0.4M, 0.5M, 0.75M, 1M or more. Non-permissive conditions can include, for example, a higher temperature (e.g., a temperature significantly higher than the Tm of the hybrid formed between the first nucleotide sequence of the primer containing the handle and the second nucleotide sequence) and / or a low level or absence of salt levels (e.g., a NaCl concentration of 0.2M, 0.1M, 0.05M, 0.001M or less).

[0087] In some embodiments, in performing Fig.12After the implantation (or pre-implantation) method shown, the implanted vector is enriched by separation from other reaction components. In one enrichment method, the implanted vector is indirectly captured by a linker portion (e.g., biotin) attached to a double-stranded target template handle on the vector. Beads having a portion (e.g., streptavidin) that in turn binds or is attached to a linker attached to a target template nucleic acid can be used to enrich the implanted vector. For example, Fig.13 As depicted, an oligonucleotide complementary to a handle sequence on a template target nucleic acid bound to a carrier and connected to a linker portion (e.g., a biotin adduct) (i.e., a "capture" oligonucleotide) is contacted with an implanted carrier and a bead (e.g., a magnetic bead) having a surface coated with streptavidin. This results in indirect capture of a double-stranded target template nucleic acid bound to a carrier by forming a bead assembly. In the case of magnetic beads, the assembly can be precipitated on a magnet, and the supernatant containing any other reaction components is removed. In some embodiments, the capture process is performed under conditions where a first nucleotide sequence and a second nucleotide sequence that are at least partially complementary to a primer containing a free handle will hybridize, such as permissive conditions. Under such conditions, the primer containing a free handle present will be in a hairpin configuration that will not hybridize with the capture oligonucleotide. Therefore, the primer containing a free handle will not be captured by the streptavidin-coated magnetic beads.

[0088] Fig.13 The process of connecting the template nucleic acid bound to the carrier to the magnetic beads is called the indirect capture method, while Fig.11 The process described in the previous section of the invention of connecting the template nucleic acid bound to the carrier to the magnetic beads is called the indirect capture method and is called the direct capture method. Fig.11 As shown, streptavidin-coated magnetic beads are bound to a biotin linker moiety that is directly attached to a strand of the target template duplex that hybridizes to a strand that is an extension primer immobilized on a support. Fig.13In the indirect capture method shown, streptavidin-coated magnetic beads are bound to biotinylated capture oligonucleotides that are not chains of the target template duplex bound to the carrier, but are hybridized to the extended single-stranded handle. From the duplex. Therefore, the bead assembly formed after the implanted carrier is captured by the magnetic beads in the direct and indirect capture methods is different. In some cases, the difference in the bead assembly may affect the association between the hybrid chains of the target template duplex bound to the carrier. For example, the force applied to the template chain directly connected to the magnetic beads by the movement of the magnetic beads may sometimes be opposite to the force applied to the duplex chain by the movement of the implanted carrier. Compared with the template duplex in which the magnetic beads are indirectly connected to the template duplex by the capture oligonucleotide, this force may have a greater effect on the related template duplex chain. The extent of the effect may be affected by one or more factors, such as the length of the template hybrid, the salt concentration of the culture medium, the temperature, and the amount of mixing, transfer, etc. of the bead assembly during capture and enrichment. As the effect of this force on the target template duplex bound to the carrier increases, the possibility of template duplex dissociation will also increase. The dissociation of the target template duplex during the enrichment process may lead to a reduction in the yield of the vector implanted by enrichment and / or an increase in the percentage of enrichment of longer template duplexes. However, the short hybridization between the handle portion of one of the template duplexes of the capture oligonucleotide and the bead assembly formed in the indirect capture method may be easier and faster to dissociate than the template duplex, thereby releasing the magnetic beads and eliminating the magnetic beads forcing the template duplex. In addition, the short nucleic acid sequence of the capture oligonucleotide and the handle portion of the template duplex may be easier to combine than the separated longer template duplex, which can alleviate any potential reduction in the yield of the vector implanted by enrichment. Therefore, capture methods and capture conditions can be selected to match the required template duplex length of the implant and the vector yield of the implant. Generally, in order to optimize the yield of the implanted vector when using the direct capture method, the bead assembly produced by such a method is processed using milder conditions (such as slow pipetting and gentle mixing), rather than violent conditions (such as vortex). When the bead assembly is produced using the indirect capture method, the carrier yield of the implant is usually not adversely affected by more violent processing of the bead assembly.

[0089] The captured implanted vector is then eluted from the capture oligonucleotide bound to the magnetic beads by denaturing the relatively small hybridization region between the capture oligonucleotide and the handle sequence on the target template under conditions in which the double-stranded target template nucleic acid bound to the implanted vector is not denatured. For example, the difference in the melting temperature (Tm) of the short hybrid formed by the handle sequence and the capture oligonucleotide (e.g., Tm in a high salt buffer is about 32°C) and the double-stranded target template nucleic acid bound to the vector (e.g., Tm greater than 32°C). For example, in some embodiments, any moderate denaturing conditions, such as increased temperature (e.g., gentle heating to greater than 35°C, such as 42°C for about 5 minutes), lower salt concentrations (e.g., low TE, addition of water) and / or physical disturbance or agitation (e.g., vortexing, pipetting) can be used to dissociate the capture oligonucleotide-handle sequence hybrid without destroying the double-stranded vector-bound target template nucleic acid. The magnetic beads bound to the capture oligonucleotide are discarded, leaving the enriched double-stranded target template bound to the implanted vector. In embodiments where the first nucleotide sequence and the second nucleotide sequence of the primer containing the handle are not complementary and do not hybridize to form a hairpin structure under any conditions used in the implantation process, an excess of capture oligonucleotides and an excess of streptavidin-coated magnetic beads can be included in the capture process to ensure capture of all template nucleic acid implanted vectors. In further downstream processing (e.g., in loading the implanted vector into a reaction site, such as a microwell on a surface, such as a chip, for sequencing a template bound to a solid support), any primer containing the handle and not bound to the support can be captured by the magnetic beads and eluted from the implanted support together with the implanted support to separate the implanted amplification reaction products from the implanted support, as described herein.

[0090] and Fig.11 Compared to the enrichment method described in , single-stranded template bound to the vector is generated after denaturation to remove the magnetic beads. Fig.13 The enrichment methods described in generate double-stranded templates bound to the deformed carrier to remove the magnetic beads. In some embodiments, attaching the double-stranded template to the carrier facilitates the detection of errors that may occur during the implantation process, eliminating consideration of any downstream analysis results of the carrier implanted with the template containing the error, thereby reducing errors, for example, in the overall results of sequencing of multiple implanted template populations. For example, in some cases, polymerization errors may occur during template-dependent extension of primers immobilized on the carrier when generating double-stranded target templates bound to the carrier. If this type of error occurs, and subsequent capture and enrichment of the implanted carrier will only produce single-stranded implanted templates, such as Fig.11As described above, the only template sequence available for downstream processes (e.g., in templated amplification and sequencing) will contain errors. In this case, the templates amplified from the implanted vector will be a monoclonal sequence population containing errors, and sequencing of the templates will produce identical reads. In contrast, if the double-stranded implanted template is generated by indirect capture and enrichment, such as Fig.13 As shown, the two chains, i.e., one is the extended fixed primer with errors and the other is the fixed primer without errors, can be used for further processing and analysis. This effectively dilutes the template error from 100% (in the case of only single-stranded implanted templates) to about 50% (i.e., the implantation error rate is reduced by about 50%). Because the template amplified from such a double-stranded implanted vector will produce a polyclonal sequence population (i.e., some sequences (about 50%) have errors, while some sequences (about 50%) have no errors), the errors can be detected in downstream analysis, such as sequencing. When analyzing the sequence results of multiple nucleic acid template populations, sequence reads from the sequencing of the polyclonal population can be excluded. Therefore, if there are no other errors in the templates bound to other vectors being sequenced, the sequencing error rate of the remaining templates is actually 0%.

[0091] In some embodiments, according to any of the pre-implantation and / or template methods provided herein (including, for example, Figure 4-7 1 and 12) produced by a series of reactions depicted in Figure 12) and the target nucleic acid attached to the carrier (enriched or not enriched) is sequenced, for example, in a massively parallel sequencing reaction. In some embodiments, the carrier (enriched or not enriched) attached to the target nucleic acid molecule is placed on an array of reaction chambers coupled to a field effect transistor (FET) or an ion-sensitive field effect sensor (ISFE), and the target nucleic acid molecule is sequenced.

[0092] In certain embodiments, template nucleic acid molecules are derived from samples from natural or non-natural sources. In certain embodiments, nucleic acid molecules in samples are derived from living organisms or cells. Any nucleic acid molecules can be used, for example, samples can include genomic DNA covering part or all of the genome, mRNA or miRNA from living organisms or cells. In other embodiments, template nucleic acid molecules are synthetic or recombinant. In certain embodiments, samples contain nucleic acid molecules with substantially the same sequence or with mixtures of different sequences. Illustrative embodiments are usually carried out using nucleic acid molecules produced in living cells and produced by living cells. Such nucleic acid molecules are typically directly from natural sources, such as cell or body fluid separation, without any in vitro amplification. Therefore, sample nucleic acid molecules are directly used in subsequent steps. In certain embodiments, nucleic acid molecules in samples can include two or more nucleic acid molecules with different sequences.

[0093] Various methods for preparing template nucleic acid molecules from samples are known in the art and can be used in any aspect of pre-implantation and / or templated methods, and systems, compositions, kits and / or devices. In some embodiments, nucleic acid molecules are present in the sample in fragmented or unfragmented form. In any of the disclosed embodiments, the nucleic acid molecules in the sample are fragmented, or further fragmented to produce nucleic acid molecules with any selected length before being used in the pre-implantation reaction. A person skilled in the art will recognize methods for performing such fragmentation to obtain fragments within a selected length range. For example, nucleic acid molecules can be fragmented using the following methods: physical methods, such as ultrasonic treatment; enzymatic methods, such as digestion by DNase I or restriction endonucleases; or chemical methods, such as heating in the presence of divalent metal cations. In some embodiments, nucleic acid molecules in the sample are fragmented to produce nucleic acid molecules with any selected length. A person skilled in the art will recognize methods for performing such fragmentation to achieve a range of selected lengths. In other embodiments, nucleic acid fragments within a range selected length are selected using methods known in the art. In some aspects, nucleic acid molecules or nucleic acid fragments of a specific size range are selected using methods known in the art. In some embodiments, the nucleic acid molecule or fragment is between about 2 and 10,000 nucleotides in length, eg, between about 2 and 5,000 nucleotides in length, between about 2 and 3,000 nucleotides in length, or between about 2 and 2,000 nucleotides in length.

[0094] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits and apparatus, and nucleic acid molecules from samples are used as part of a high-throughput sequencing workflow, such as to generate a library of template nucleic acid molecules. In some embodiments, a population of different template nucleic acid molecules amplified using any of the amplification methods taught in the present invention comprises a library of template nucleic acid molecules having a nucleic acid adapter sequence at one or both ends. For example, the template nucleic acid molecules in the library may comprise a first and a second end, wherein the first end is joined to a first nucleic acid adapter. The template nucleic acid molecules in the library may also comprise a second end joined to a second nucleic acid adapter. The first and second nucleic acid adapters may be joined or otherwise introduced into the template nucleic acid molecules. The adapters may be joined or otherwise introduced into the ends of the linear template or into the body of the linear or circular template nucleic acid molecules. Optionally, the template nucleic acid molecules may be circularized after joining or introducing the adapters. In some embodiments, the first adapter is joined or introduced into the first end of the linear template, and the second adapter is joined or introduced into the second end of the template. The first and second adapters may have the same or different sequences. The first and second adapters may have the same or different primer binding sequences. In some embodiments, at least a portion of the first or second nucleic acid adaptor (ie, a portion of a template nucleic acid molecule in a library) can hybridize to a first primer, which is a universal primer.

[0095] Nucleic acid molecules may have 5' and / or 3' overhangs that can be repaired before further library preparation. In illustrative embodiments, template nucleic acid molecules with 5' and 3' overhangs are repaired using methods known in the art to produce blunt-ended sample nucleic acid molecules. For example, in an appropriate buffer, the polymerase and exonuclease activity of Klenow large fragment polymerase can be used to fill in 5' overhangs and remove 3' overhangs on nucleic acid molecules. In some embodiments, phosphate is added to the 5' end of the repaired nucleic acid molecule using polynucleotide kinase (Polynucleotide Kinase; PNK) and reaction conditions that will be understood by those skilled in the art. In other illustrative embodiments, a single nucleotide or multiple nucleotides are added to one chain of a double-stranded molecule to produce a "sticky end". For example, adenosine (A) can be connected to the 3' end of a nucleic acid molecule (adding an A tail). In some embodiments, other sticky ends other than the A overhang can be used. In some embodiments, other adapters are added, such as circular junction adapters. In some embodiments, adapters are added during the PCR step. In any one embodiment of the present teachings, all or any combination of these modifications are not performed, performed, or performed. Many kits and methods for generating a population of template nucleic acid molecules for subsequent sequencing are known in the art. Such kits will typically be modified to include adaptors customized for the amplification and sequencing steps of the methods and compositions of the present teachings. Adaptor ligation may also be performed using commercially available kits, such as the ligation kit present in the Agilent SureSelect kit (Agilent).

[0096] In certain embodiments, amplification method is optionally included in the target enrichment step before, during or after library preparation and before pre-implantation reaction. Can be enriched for example by multiple nucleic acid amplification or hybridization to comprise the target nucleic acid molecule of target locus or region of interest. Carrying out multiple nucleic acid amplification to produce the various methods (such as multiple PCR) of amplicon is known in the art, and can be used in any one embodiment of the content of the present invention. Before adding template nucleic acid molecule in pre-implantation reaction mixture, can be enriched by any method, then carry out universal amplification reaction. Any embodiment of the present teachings comprises enriching for a plurality of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 target nucleic acid molecules, target loci, or regions of interest. In any of the disclosed embodiments, the target locus or region of interest is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides long and comprises a portion or all of a template nucleic acid molecule. In other embodiments, the target locus or region of interest is between about 1 and 10,000 nucleotides in length, such as between about 2 and 5,000 nucleotides in length, between about 2 and 3,000 nucleotides in length, or between about 2 and 2,000 nucleotides in length. In any embodiment of the present teachings, multiplex nucleic acid amplification comprises generating at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 copies of each target nucleic acid molecule, target locus, or region of interest. In any of the disclosed embodiments, methods for preimplantation, and related compositions, systems, kits, and apparatuses, comprise generating a preimplanted population of solid supports using amplicons from multiplexed nucleic acid amplification in a preimplantation reaction.

[0097] In some embodiments, after library preparation and optional enrichment steps, the template nucleic acid molecule library is templated onto one or more carriers. In some embodiments, one or more carriers are templated in two or more reactions, and the pre-implantation reaction is to produce a pre-implantation carrier and one or more (e.g., two) templated reactions connected with template nucleic acid molecules using one or more pre-implantation carrier amplification. In some embodiments, the pre-implantation (or implantation) reaction includes hybridization of a template nucleic acid (e.g., a single-stranded template nucleic acid) with a primer oligonucleotide fixed on a carrier, and the carrier is complementary to the sequence contained in the template nucleic acid. In some embodiments, the pre-implantation (or implantation) reaction is further included in the template nucleic acid and the primer hybridization fixed on the carrier before and / or at the same time the library or sample nucleic acid is operated. Such manipulation includes nucleic acid amplification. As used herein, nucleic acid amplification refers to the process of synthesizing a new chain of nucleic acid by nucleotide polymerization, and involves one or more cycles of the following: separation of double-stranded nucleic acid, such as denaturation or dissociation into single strands, hybridization of primers and separated single strands of double-stranded nucleic acids, and extension of hybridization primers. In some embodiments, the pre-implantation reaction includes nucleic acid amplification and can be performed using a variety of methods that the technician will understand. For example, in some embodiments, the pre-implantation reaction can be carried out in an RPA reaction, a template walking reaction, or a PCR. In the RPA reaction, in the presence of primers and nucleotides, a recombinase, a polymerase, and optionally a recombinase auxiliary protein are used to amplify the template nucleic acid molecule. The recombinase and optionally the recombinase auxiliary protein can dissociate at least a portion of the double-stranded template nucleic acid molecules so that the primers hybridize, and then the polymerase can bind to start replication. In some embodiments, the recombinase auxiliary protein is a single-stranded binding protein (SSB) that prevents the dissociated template nucleic acid molecules from hybridizing again. Typically, the RPA reaction is carried out at an isothermal temperature. In the template walking reaction, in the presence of primers and nucleotides, a polymerase is used to amplify the template nucleic acid molecules. In PCR, the double-stranded template nucleic acid molecules are dissociated by thermal cycling. After cooling, the primer binds to the complementary sequence and can be used for the replication of the polymerase. In some embodiments of the embodiments provided herein, the pre-implantation reaction is performed in a pre-implantation reaction mixture, which is formed by the components necessary for amplifying the template nucleic acid molecules. In any of the disclosed aspects, the pre-implantation reaction mixture comprises some or all of the following: a population of template nucleic acid molecules, a polymerase, one or more carriers (e.g., solid carriers) with a first primer population connected, nucleotides, and cofactors (such as divalent cations). In some embodiments, the pre-implantation reaction mixture further comprises a second primer and optionally a diffusion-restricting agent.In some embodiments, the template nucleic acid molecule population includes template nucleic acid molecules joined to at least one adapter sequence hybridized to the first or second primer. In some embodiments, such as in emulsion RPA or emulsion PCR, the reaction mixture forms an emulsion. In the pre-implantation reaction carried out by the RPA reaction, the pre-implantation reaction mixture includes a recombinase and optionally a recombinase auxiliary protein. The various components of the reaction mixture are further discussed in detail herein.

[0098] In some embodiments, the pre-implantation reaction mixture comprises a template nucleic acid molecule population that is generally derived from library preparation or target enrichment. In some embodiments, the template nucleic acid molecule or template nucleic acid molecule population is at least some and generally all members of a template nucleic acid molecule library. In some embodiments, the pre-implantation reaction mixture comprises at least one template nucleic acid molecule. In some embodiments, the pre-implantation reaction mixture comprises at least two template nucleic acid molecules with different sequences. In illustrative embodiments, the pre-implantation reaction mixture comprises a template nucleic acid molecule population with different sequences. In some embodiments, the pre-implantation reaction mixture comprises a substantially monoclonal template nucleic acid molecule population. In any one embodiment of the present invention, when the template nucleic acid molecule is alternatively referred to herein, the template nucleic acid molecule is a polynucleotide, (the template nucleic acid molecule is interchangeably referred to herein as a template or nucleic acid template or a polynucleotide template). In various embodiments, the template nucleic acid molecule is a polymer of deoxyribonucleotides, ribonucleotides and / or its analogs. In some embodiments, the polynucleotide is a naturally occurring, synthetic, recombinant, cloned, amplified, unamplified or archived (e.g., preserved) form. In some embodiments, the polynucleotide is a DNA, cDNA, RNA or chimeric RNA / DNA and a nucleic acid analog.

[0099] The template nucleic acid molecule to be amplified can be double-stranded, or it is at least partially double-stranded using appropriate procedures before the pre-implantation reaction. In some embodiments, the template is a straight chain. Alternatively, the template can be circular, or a combination of straight and circular regions. In some embodiments, amplification includes forming a partially denatured or dissociated template or a completely denatured or dissociated template. For example, amplification may include partially denaturing the double-stranded template nucleic acid molecule. Optionally, partially denaturing includes subjecting the double-stranded template nucleic acid molecule to partially denaturing conditions. In some embodiments, the partially denatured template includes a single-stranded portion and a double-stranded portion. In some embodiments, the single-stranded portion includes a first primer binding sequence. In some embodiments, the single-stranded portion includes a second primer binding sequence. In some embodiments, the single-stranded portion includes a first primer binding sequence and a second primer binding sequence.

[0100] Optionally, the double-stranded template nucleic acid molecule comprises a forward strand. The double-stranded template nucleic acid molecule may further comprise a reverse strand. The forward strand optionally comprises a first primer binding sequence. The reverse strand optionally comprises a second primer binding sequence. As disclosed above, typically during library preparation, primer binding sequences are connected. In certain embodiments, the template nucleic acid molecule already comprises a first primer binding sequence and optionally a second primer binding sequence. Alternatively, the template nucleic acid molecule optionally does not initially comprise a primer binding sequence, and library preparation may optionally comprise connecting or introducing a primer binding sequence to a template, as disclosed above.

[0101] In some embodiments, the template nucleic acid molecule comprises a single-stranded or double-stranded polynucleotide, or a mixture of the two. In some embodiments, the template nucleic acid molecule comprises a polynucleotide having the same or different nucleotide sequences. In some embodiments, the template nucleic acid molecule comprises a polynucleotide having the same or different lengths. In various embodiments, the pre-implantation reaction mixture comprises about 2 and 10 12 different template nucleic acid molecules, for example, between about 2 and 10 11 different template nucleic acid molecules, between about 2 and 10 10 different template nucleic acid molecules, between about 2 and 10 9 different template nucleic acid molecules, between about 2 and 10 8 different template nucleic acid molecules, between about 2 and 10 7 different template nucleic acid molecules, between about 2 and 10 6 In any of the disclosed embodiments, the pre-seeded reaction mixture or the templated reaction mixture comprises between 5×10 6 with 10 10 For example, the pre-implantation reaction mixture or the template reaction mixture may contain 5×10 8 With 10×10 9 The carrier between 5×10 8 With 9×10 9 The carrier between 5×10 8 With 8×10 9 The carrier between 5×10 8 With 7×10 9 The carrier between 5×10 8 With 6×10 9 The carrier between 5×10 8 With 5×10 9 The carrier between 5×10 8 With 4×10 9 The carrier between 5×108 With 3×10 9 The carrier between 5×10 8 With 2×10 9 The carrier between 5×10 8 with 10 9 The carrier between 10 9 with 10 10 The carrier between 10 9 With 9×10 9 The carrier between 10 9 With 8×10 9 The carrier between 10 9 With 7×10 9 The carrier between 10 9 With 6.5×10 9 The carrier between 10 9 With 6×10 9 The carrier between 10 9 With 5.5×10 9 The carrier between 10 9 With 5×10 9 The carrier between 10 9 With 4.5×10 9 Between the vectors or in 10 9 With 4×10 9 In another example, the pre-implantation reaction mixture or the template reaction mixture may contain at least about 5×10 8 carriers, at least about 10 9 carriers, at least about 2×10 9 carriers, at least about 3×10 9 carriers, at least about 4 × 10 9 carriers, at least about 5×10 9 carriers, at least about 5.5×10 9 carriers, at least about 6×10 9 carriers, at least about 6.5×10 9 carriers, at least about 7×10 9 carriers, at least about 7.5×10 9 carriers, at least about 8×10 9 carriers, at least about 8.5×10 9 carriers, at least about 9×10 9 carriers, at least about 9.5×10 9 vectors or at least about 10 10The solid support may have a minimum cross-sectional length (e.g., diameter) of 50 microns or less, preferably 10 microns or less, 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3, or 0.4 microns or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers). In any embodiment of the present teachings, the volume of the pre-implantation reaction mixture and / or the templated reaction mixture is between about 50 and 2,000 μl, such as between about 50 and 1,500 μl, between about 50 and 1,000 μl, between about 50 and 500 μl, between about 50 and 250 μl, or between about 50 and 150 μl.

[0102] Preimplantation reaction is carried out to produce one or more preimplanted carriers. Therefore, in any disclosed aspect, the preimplantation reaction mixture may include one or more solid or semi-solid carriers connected to the template nucleic acid molecule in the preimplantation reaction. As used herein, a solid or semi-solid carrier may also refer to a carrier with a connection site that can be clearly analyzed in a downstream sequencing method. In an illustrative embodiment, one or more carriers include substantially the same first primer population connected. In certain embodiments, at least one template nucleic acid molecule in the reaction mixture includes a first primer binding sequence. The first primer binding sequence may be substantially the same or substantially complementary to the sequence of the first primer. In certain embodiments, at least one, some or all of the carriers include substantially the same first primer populations. In certain embodiments, all primers on the carrier are substantially the same as each other, or all include substantially the same first primer sequence. In certain embodiments, at least one of the carriers includes two or more different primers connected thereto. For example, at least one carrier may include a first primer population and a second primer population. The carrier may be connected to a universal primer. Universal primers are optionally hybridized (or capable of hybridizing) with all or substantially all template nucleic acid molecules in the reaction mixture. The reaction mixture may comprise a first carrier covalently linked to a first target-specific primer and a second carrier covalently linked to a second target-specific primer, wherein the first and second target-specific primers are different from each other. Optionally, the first target-specific primer is substantially complementary to the first target nucleic acid sequence, and the second target-specific primer is substantially complementary to the second target nucleic acid sequence, and wherein the first and second target nucleic acid sequences are different.

[0103] In some embodiments, two or more different template nucleic acid molecules with the first primer binding sequence are included in the pre-implantation reaction mixture. In some embodiments, at least two different template nucleic acid molecules are directly amplified onto a carrier, such as a site on a carrier comprising multiple sites, a bead or microparticle, or a reaction chamber of an array. Template nucleic acid molecules can be pre-implanted in a large number in a solution, and subsequently distributed to a hole array or a reaction site on a solid carrier. In some embodiments, a hole or reaction site contains a pre-implanted carrier of each hole or reaction site. In some embodiments, the template nucleic acid connected to the pre-implanted carrier distributed to the hole or reaction site then undergoes one or more templated reactions, wherein the template is amplified on the carrier to produce a substantially monoclonal population of template nucleic acid. Alternatively, a solid carrier can be distributed into a hole array, and the template nucleic acid molecules can be pre-implanted on the solid carrier while being maintained in situ in the hole array. In some embodiments, the template nucleic acid connected on the carrier pre-implanted into the hole or reaction site then undergoes one or more templated reactions, wherein the template is amplified on the carrier to produce a substantially monoclonal population of template nucleic acid. In some embodiments, the method for nucleic acid amplification comprises one or more surfaces.

[0104] In some embodiments, the surface is connected to a first primer population, and the first primers in the population share a common first primer sequence. In some embodiments, the surface is connected to a first primer population and a second primer population, and the first primers in the population share a common first primer sequence, and the second primers in the second primer population share a common second primer sequence. In some embodiments, the first primer population is fixed on the surface. In other embodiments, the first primer population and the second primer population are fixed on the surface.

[0105] The carrier or surface can be coated with acrylamide, carboxylic acid or amine compounds for connecting nucleic acid molecules (e.g., the first primer or the second primer). In some embodiments, amino-modified nucleic acid molecules (e.g., primers) are connected to a carrier coated with carboxylic acid. In some embodiments, amino-modified nucleic acid molecules are reacted with EDC (or EDAC) to connect to a surface (with or without NHS) coated with carboxylic acid. The first primer can be connected to an acrylamide compound coated on the surface. The particles can be coated with avidin-like compounds (e.g., streptavidin) to bind biotin-labeled nucleic acids.

[0106] In some embodiments, the reaction mixture comprises a plurality of different surfaces, for example, the pre-implantation reaction mixture comprises one or more beads (e.g., particles, nanoparticles, microparticles, and the like), and at least two different template nucleic acid molecules are amplified onto different beads, thereby forming at least two different beads, each of which is attached to a different template nucleic acid molecule. In some embodiments, the pre-implantation reaction mixture comprises a single surface (e.g., a planar surface, a flow cell, or an array of reaction chambers), and at least two different template nucleic acid molecules are amplified onto two different regions, sites, or locations on the surface, thereby forming a single surface attached to two or more template nucleic acid molecules.

[0107] In some embodiments, the surface of the solid support is porous, semi-porous or non-porous. In some embodiments, the surface is a planar surface, as well as a concave surface, a convex surface or any combination thereof. In some embodiments, the surface is the inner wall of a bead, a particle, a microparticle, a sphere, a filter, a flow cell, a hole, a groove, a channel reservoir, a gel or a capillary. In some embodiments, the surface comprises a texture (e.g., etching, forming a cavity, a hole, a three-dimensional scaffold or a bump).

[0108] In some embodiments, the surface of the solid support is magnetic or paramagnetic beads (e.g., magnetic or paramagnetic nanoparticles or microparticles). In some embodiments, the paramagnetic microparticles are paramagnetic beads linked to streptavidin (e.g., Dynabeads from Invitrogen, Carlsbad, CA). TM M-270). The particles may have an iron core, or may be a hydrogel or agarose (e.g., Sepharose TM ).

[0109] In some embodiments, the surface comprises the surface of a bead. In some embodiments, the bead is a polymer material. For example, the bead can be a gel, a hydrogel, or an acrylamide polymer. The bead can be porous. The particle can have holes or pores, or can comprise a three-dimensional structure. In some embodiments, the particle is an Ion Sphere. TM particles (Thermo Fisher Scientific, Waltham, MA).

[0110] Typically, the polymer particle or bead carrier can be treated to contain a biomolecule, including nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, sugars, polysaccharides, lipids or derivatives or analogs thereof. For example, the polymer particle can be bound or linked to the biomolecule. The end or any internal portion of the biomolecule can be bound or linked to the polymer particle. The polymer particle can be bound or linked to the biomolecule using a linking chemistry. Linking chemistry includes covalent or non-covalent bonds, including ionic bonds, hydrogen bonds, affinity bonds, dipole-dipole bonds, van der Waals bonds, and hydrophobic bonds. The ligation chemistry method includes affinity between binding partners, such as affinity between: an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or an immunoreactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigenin antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement.

[0111] Specifically, a solid phase carrier, such as a bead carrier, may contain copies of polynucleotides. In a particular example, polymer particles may be used as carriers of polynucleotides during sequencing techniques. For example, such hydrophilic particles may be fixed to polynucleotides sequenced using fluorescent sequencing techniques. In another example, hydrophilic particles may be fixed to multiple copies of polynucleotides sequenced using ion sensing techniques. Alternatively, the above-described treatment may improve the bonding of polymer matrix to the sensor array surface. The polymer matrix may capture analytes, such as polynucleotides for sequencing.

[0112] In some embodiments, one or more nucleic acid templates are fixed to one or more carriers. Template nucleic acid molecules can be fixed to the carrier by any method, including but not limited to physical adsorption, by ionic or covalent bond formation or a combination thereof. The solid carrier can comprise a polymer, glass or metal material. Examples of solid carriers include membranes, planar surfaces, microtiter plates, beads, filters, test strips, slides, coverslips and tubes. A solid carrier means any solid phase material on which an oligomer is synthesized, connected, joined or otherwise fixed. The carrier may optionally comprise "resin", "phase", "surface" and "carrier". The carrier may include organic polymers such as polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyethyleneoxy and polyacrylamide and copolymers and grafts thereof. The carrier may also be inorganic, such as glass, silica, controlled-pore-glass (CPG) or reversed-phase silica. The configuration of the carrier may be in the form of beads, spheres, particles, particles, gels or surfaces. The surface may be planar, substantially planar or non-planar. The carrier can be porous or non-porous, and can have swelling or non-swelling characteristics. The carrier can be molded to contain one or more holes, depressions or other containers, vessels, features or positions. One or more carriers can be configured in different positions in the array. The carrier is optionally addressable (e.g., for mechanical delivery of reagents), or by detection means, including addressing using laser irradiation scanning and confocal or polarized focusing. The carrier (e.g., bead) can be placed in or on another carrier (e.g., in the hole of a second carrier). In certain embodiments, the carrier is an ionic spherical particle.

[0113] In some embodiments, the solid support is a "microparticle", "bead", "microbead", etc. (optionally but not necessarily spherical in shape) having a minimum cross-sectional length (e.g., diameter) of 50 microns or less, preferably 10 microns or less, 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3, or 0.4 microns or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers). Micron particles (e.g., Dynabead, Dynal, Oslo, Norway) can be made of various inorganic or organic materials, including but not limited to glass (e.g., controlled pore glass), silica, zirconium oxide, cross-linked polystyrene, polyacrylate, polymethyl methacrylate, titanium dioxide, latex, polystyrene, etc. Magnetization can facilitate aggregation and concentration of microparticle-attached reagents (e.g., polynucleotides or ligases) after amplification, and can also facilitate additional steps (e.g., washing, reagent removal, etc.). In certain embodiments, populations of microparticles having different shapes sizes and / or colors are used. The microparticles may optionally be encoded, for example with quantum dots, so that each microparticle or group of microparticles may be individually or uniquely identified.

[0114] In some embodiments, the bead surface is functionalized for connecting the first primer population. In some embodiments, the bead is any size that can be placed in a reaction chamber. For example, a bead can be placed in a reaction chamber. In some embodiments, more than one bead is placed in a reaction chamber. In some embodiments, the minimum cross-sectional length (e.g., diameter) of the bead is about 50 microns or less, or about 10 microns or less, or about 3 microns or less, about 1 micron or less, about 0.5 micron or less, such as about 0.1, 0.2, 0.3 or 0.4 microns or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers).

[0115] In some embodiments, before the pre-implantation reaction, two or more different template nucleic acid molecules are positioned, placed or placed in different sites. In some embodiments, two or more different template nucleic acid molecules are pre-implanted in a solution, optionally in a single pre-implantation reaction mixture, and then after such amplification, the resulting two or more substantially monoclonal template nucleic acid molecule populations are positioned, placed or placed in different sites. Different sites are optionally members of array sites. The array may include a two-dimensional site array on a surface (e.g., a flow cell, an electronic device, a transistor chip, a reaction chamber, a channel, and the like), or a three-dimensional site array in a matrix or other medium (e.g., solid, semi-solid, liquid, fluid, and the like).

[0116] In some embodiments, the method for pre-implanting the template nucleic acid molecule into one or more carriers and the templated reaction generally use one or more enzymes capable of polymerization. In any one embodiment of the present invention, the one or more enzymes capable of polymerization include at least one polymerase. In some embodiments, the at least one polymerase includes a thermostable or thermolabile polymerase. In some embodiments, the at least one polymerase includes a DNA or RNA polymerase that maintains sufficient catalytic activity to polymerize or incorporate at least one nucleotide under any suitable conditions. In various embodiments, the at least one polymerase includes a mutant DNA or RNA polymerase that maintains sufficient catalytic activity to polymerize nucleotides under any suitable conditions. In various embodiments, the at least one polymerase includes one or more amino acid mutations that maintain sufficient catalytic activity to polymerize. The polymerase may optionally have or lack exonuclease activity. In some embodiments, the polymerase has 5' to 3' exonuclease activity, 3' to 5' exonuclease activity, or two. Optionally, the polymerase lacks any one or more of such exonuclease activities. In some embodiments, the polymerase has strand displacement activity. Examples of applicable strand displacement polymerases include bacteriophage Φ29 DNA polymerase and Bst DNA polymerase.

[0117] In some embodiments, the polymerase comprises any enzyme or its fragment or subunit that can catalyze the polymerization of nucleotides and / or nucleotide analogs. In some embodiments, the polymerase requires an extendable 3' end. For example, the polymerase requires the terminal 3'OH of the nucleic acid primer to start nucleotide polymerization. The polymerase can be other than a thermostable polymerase. For example, the polymerase can be active at 37°C, and / or more active at 37°C than at 50°C, 60°C, 70°C or higher temperatures. In some embodiments, the polymerase can be active at 40°C, and / or more active at 40°C than at 50°C, 60°C, 70°C or higher temperatures. In various embodiments, the polymerase can be active at 42°C, 45°C, 50°C, 55°C or 60°C, and / or more active at these temperatures than at 37°C.

[0118] The polymerase may include any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid chain. Typically, but not necessarily, such nucleotide polymerization can be performed in a template-dependent manner. In some embodiments, the polymerase is a high-fidelity polymerase. Such polymerases may include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase is a mutant polymerase having one or more mutations, wherein the one or more mutations involve replacement of one or more amino acids with other amino acids, insertion or deletion of one or more amino acids from a polymerase, or connection of two or more parts of the polymerase. As used herein, the term "polymerase" and its variations also refer to a fusion protein comprising at least two parts connected to each other, wherein the first part may include a peptide that can catalyze the polymerization of nucleotides into a nucleic acid chain and is connected to the second part, and the second part may include a second polypeptide, such as a reporter enzyme or a domain that enhances processivity. Typically, the polymerase comprises one or more active sites that can perform nucleotide binding and / or nucleotide polymerization catalysis. In some embodiments, the polymerase may comprise or lack other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase is isolated from a cell or produced using recombinant DNA technology or chemical synthesis methods. In any one embodiment of the present invention, the polymerase is expressed in a prokaryotic, eukaryotic, viral or bacteriophage organism. In various embodiments, the polymerase is a DNA polymerase and includes but is not limited to bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases and bacteriophage DNA polymerases. In various embodiments, the expressed polymerase is purified using methods known in the art. In some embodiments, the polymerase is a post-translationally modified protein or fragment thereof.

[0119] In some embodiments, the polymerase comprises any one or more polymerases or biologically active fragments of a polymerase as described in U.S. Patent Publication No. 2011 / 0262903, published by Davidson et al. on October 27, 2011; and / or International PCT Publication No. WO 2013 / 023176, published by Vander Horn et al. on February 14, 2013, which are incorporated herein by reference in their entirety.

[0120] In some embodiments, the polymerase is a replicase, a DNA-dependent polymerase, an initiator, an RNA-dependent polymerase (including an RNA-dependent DNA polymerase, such as a reverse transcriptase), a thermolabile polymerase, or a thermostable polymerase. In some embodiments, the polymerase is any A or B family type polymerase. Many types of A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C (e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., human Pol β), and Y family (e.g., E. coli UmuC / DinB and eukaryotic RAD30 / xeroderma pigmentosum variants) polymerases are described in Rothwell and Watson 2005 Advances in Protein Chemistry 71:401-440. In some embodiments, the polymerase is a T3, T5, T7, or SP6 RNA polymerase.

[0121] In some embodiments, a nucleic acid amplification reaction is performed using a single type of polymerase and / or ligase, or a mixture of polymerases and / or ligases. In some embodiments, a nucleic acid amplification reaction is performed using a low-fidelity or high-fidelity polymerase, or fidelity is not considered.

[0122] An exemplary polymerase is Bst DNA polymerase (Exonuclease Minus), which is a 67 kDa Bacillus stearothermophilus DNA polymerase protein (large fragment) exemplified in Accession No. 2BDP_A, which has 5' to 3' polymerase activity and strand displacement activity but lacks 3' to 5' exonuclease activity. Other polymerases include Taq DNA polymerase I from Thermus aquaticus (exemplified by Accession No. 1TAQ), Eco DNA polymerase I from Escherichia coli (Accession No. P00582), Aea DNA polymerase I from Aquifexaeolicus (Accession No. 067779), or a functional fragment or variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity at the nucleotide level, for example.

[0123] In illustrative embodiments, the DNA polymerase is Bsu DNA polymerase (Large Fragment (NEB)). The Bsu DNA polymerase I Large Fragment retains the 5' to 3' polymerase activity of Bacillus subtilis DNA polymerase I (1) but lacks the 5' to 3' exonuclease domain. In certain embodiments, the Bsu DNA polymerase Large Fragment lacks the 3' to 5' exonuclease activity. In various embodiments, the Bsu DNA polymerase Large Fragment has optimal activity at 37°C.

[0124] In certain illustrative embodiments, particularly when the pre-implantation reaction is an RPA reaction, the one or more enzymes capable of polymerizing include T5 or T7 DNA polymerase. In some embodiments, the one or more enzymes capable of polymerizing include T5 or T7 DNA polymerases having one or more amino acid mutations that reduce 3′ to 5′ exonuclease activity. In some embodiments, the T5 or T7 DNA polymerases having one or more amino acid mutations that reduce 3′ to 5′ exonuclease activity do not contain amino acid mutations that interfere with the processivity of the T5 or T7 DNA polymerase. In some embodiments, the T5 or T7 DNA polymerases include one or more amino acid mutations that eliminate detectable 3′ to 5′ exonuclease activity; and wherein the one or more amino acid mutations do not interfere with the processivity of the T5 or T7 DNA polymerase. In certain illustrative embodiments, the pre-implantation reaction mixture includes Sau polymerase, T7 DNA polymerase with reduced 3′ to 5′ exonuclease activity, Bsu polymerase, or a combination thereof. These polymerases, which are particularly suitable for RPA reactions, are not only very suitable for pre-implantation reactions, but also for templated reactions.

[0125] In some embodiments, the one or more enzymes capable of polymerizing comprise any suitable RNA polymerase. Suitable RNA polymerases include, but are not limited to, T3, T5, T7, and SP6 RNA polymerases.

[0126] In various embodiments, the template nucleic acid molecule used in any of the methods (including pre-implantation reactions and templated reactions) of the present teachings generally comprises a first primer binding sequence (e.g., "forward") and optionally a second primer binding sequence (e.g., "reverse"). Primers include any single-stranded nucleic acid molecule that can initiate nucleic acid synthesis once hybridized with a complementary nucleic acid sequence. Typically, this nucleic acid synthesis occurs in a template-dependent manner, and nucleotides are polymerized to at least one end of the primer during this nucleic acid synthesis process. As used herein, the term "primer extension" and its variants relate to any method for catalyzing nucleotide incorporation into the ends of nucleic acid molecules. In some embodiments of the methods and compositions provided herein, the pre-implantation reaction mixture and the templated reaction comprise a first primer population and optionally a second primer population that binds to a forward primer binding sequence and a reverse primer binding sequence, respectively. In some embodiments, the first and second primers are referred to as primer pairs. In some embodiments, the first primer and / or the second primer are typically universal primers. The first primer may bind to a forward primer binding sequence or a reverse primer binding sequence, and the second primer may bind to a forward primer binding sequence or a reverse primer binding sequence. Thus, the terms "first" and "second" when used herein with respect to primers are relative terms, and each may refer to a forward or reverse primer, depending on the context in which they are used.

[0127] In any one of the disclosed embodiments, the reaction mixture comprises a first primer population and a second primer population that are bound to a sequence within a template nucleic acid molecule. The first primer population can be the same copy or a different sequence. The second primer population can be the same copy or a different sequence. However, the first primer population and the optional second primer population are generally universal primers, and all copies are identical. Therefore, in illustrative embodiments, both the first primer population and the second primer population are universal primers that bind to the universal primer binding sequence on the template nucleic acid molecule. In other embodiments, the first primer population and the second primer population are both target-specific primers. The first primer population and the second primer population can have the same or different sequences. In any one of the embodiments of the present invention, before incubation with the pre-implantation reaction mixture, the first primer population and / or the second primer population are connected to one or more carriers. In other embodiments, during incubation with the pre-implantation reaction mixture, the first primer population and / or the second primer population are in solution. In illustrative embodiments, a first population of primers is attached to one or more supports prior to incubation with the preimplantation reaction mixture; and a second population of primers is typically in solution during incubation with the preimplantation reaction mixture.

[0128] Therefore, in these illustrative embodiments of the first primer population and the second primer population fixed in the solution, without being limited by theory, during the pre-implantation reaction, the template nucleic acid is at least partially denatured, and the first primer binding site on the template is combined with the first primer connected to the solid support. The polymerase uses the first primer to produce the complementary chain of one chain of the template nucleic acid. That complementary chain is now covalently linked to the solid support through the primer. The second primer in the solution is in the form of a complex with the recombinase, and is combined with the primer binding site on the complementary chain, so that the combined template nucleic acid molecule is partially denatured. The polymerase uses the primer to synthesize a new chain identical to the original template nucleic acid chain. It is believed that this chain will be partially denatured by combining the complex of the recombinase and the nearby first primer connected to the solid support, and the polymerase synthesizes another complementary chain. Through the repeated steps of this method, a substantially monoclonal population of the template nucleic acid molecule is produced during the pre-implantation reaction, and further amplified during the templated reaction.

[0129] In certain embodiments, the disclosure generally relates to methods, and systems, compositions, kits and equipment, wherein the primers generally have free 3' hydroxyl groups. In certain embodiments, the primers are polymers of ribonucleotides, deoxyribonucleotides or their analogs. In certain embodiments, the primers are naturally occurring, synthetic, recombinant, cloned, amplified or unamplified forms. In certain embodiments, the primers are included in phosphodiester bonds between all nucleotides. In any one embodiment of the present invention, the length of the primers is between about 5 and 100 nucleotides, such as a length between about 5 and 80 nucleotides, a length between about 5 and 60 nucleotides, a length between about 5 and 40 nucleotides, a length between about 10 and 75 nucleotides, a length between about 15 and 75 nucleotides or a length between about 20 and 50 nucleotides.

[0130] In certain embodiments, at least one primer has modification.For example, ribonucleotide, deoxyribonucleotide or its analog can have biotin or azide connected thereto, and in certain embodiments, it is used as linker part.In certain embodiments, ribonucleotide, deoxyribonucleotide or its analog has fluorophore, phosphorylation or spacer connected.In certain embodiments, primer is end-capped and / or is fusion primer or fusion polynucleotide, and wherein the different regions of primer or polynucleotide are designed to be combined with one of two primer binding sites and / or are designed to be combined by one of two primers.

[0131] In some embodiments, the primer is a blocked primer to prevent the 3' end of the primer from extending. In some embodiments, the blocked primer is a tailed primer, wherein the 5' end comprises a sequence that is non-complementary to the template nucleic acid molecule. This 5' sequence can be used as a template for a primer extension reaction. In some embodiments, the primer is blocked, wherein the length of the 5' domain is between 15 and 30 nucleotides. In some embodiments, the primer is included in a blocking portion at its 5' or 3' end or at the 5' and 3' ends. In reactions involving primer extension (e.g., pre-implantation amplification), the blocking portion at the 3' end of the blocked fusion primer can reduce the level of primer dimer formation. In certain embodiments, the pre-implantation reaction mixture comprises a blocked primer, wherein the length of the 3' domain is between 14 and 25 nucleotides. In other embodiments, the length of the 3' domain is between 15 and 25 nucleotides. In other embodiments, the 5' domain is at least 15 nucleotides and the 3' domain is at least 10 nucleotides, wherein the length of the primer does not exceed 100, 90, 80, 75, 70, 60 or 50 nucleotides. In an embodiment, the 3' nucleotide of the 3' domain of the forward primer is mismatched with the forward primer binding sequence. In an embodiment, the ribobase (ribobase) separating the 5' domain and the 3' domain of the end-capped primer comprises rU, rG, rC or rA. In certain embodiments, the ribobase separating the 5' domain and the 3' domain of the end-capped primer comprises rC. In any one embodiment of the present invention, the length of the 3' domain of the end-capped primer is 14 to 20 nucleotides, and the ribobase is rU, rG, rC or rA. In certain embodiments, the primer is blocked because the primer contains a non-replicable part. The non-replicable part can be any composition that cannot be replicated by a polymerase, such as a polymerase termination site. For example, such primers can have a polymerase termination site non-replicable part, which is located between the 5' end sequence of the primer that is not complementary to the template nucleic acid and the 3' end sequence of the primer that is complementary to the template nucleic acid. In this case, the template-dependent polymerization of the extension product containing such primers will not include the sequence of the 5' end of the primer in the template-dependent polymerization, because the 5' end of the primer is located after the polymerase termination site, which will prevent the polymerase from using the 5' end of the primer as a template. Such non-replicable parts include any part of the nucleotide polymerization based on the template that cannot support the polymerase to carry out. For example, the non-replicable part can include a non-nucleotide part (for example, PEG or other carbon-based spacers, amino acids or nucleotide analogs) that is not used for the polymerase recognition of primer extension. In certain embodiments, the nucleotide sequences before and after the non-replicable part in such primers can hybridize to each other under certain conditions. The example of the primer containing the non-replicable part is described in, for example, International Application Publication No. WO2014 / 062717, which is incorporated herein by reference in its entirety.

[0132] In some embodiments of the present teachings, the reaction mixture includes an enzyme that removes a portion of the blocked primer to leave a free 3'OH. ​​After removing the blocked end of the primer, the polymerase can start replication from the free 3'OH to begin replicating the template strand. In some embodiments, this enzyme is an RNase, particularly RNase H. Those skilled in the art will recognize other compositions of blocked primers used and suitable enzymes for removing a portion of the blocked primer.

[0133] In some embodiments, the primers used in the methods, kits, compositions, systems and devices provided herein have a secondary structure that inhibits hybridization of the primer with another polynucleotide (e.g., a capture oligonucleotide) under certain conditions. For example, the primer can be a primer that can form a hairpin or a stem-loop structure. Examples of such primers are described in, for example, International Application Publication No. WO2014 / 062717.

[0134] Any other amplification reaction (including templated reaction mixture) in the pre-implantation reaction mixture and the method provided herein, usually comprises nucleotides or their analog sources used as substrates for extension reactions by polymerase. In any one embodiment of the present invention, the pre-implantation reaction mixture usually comprises nucleotides (dNTPs) of a substantially monoclonal population of template nucleic acid molecule sequences connected to one or more carriers for chain extension of template nucleic acid molecules. In certain embodiments, nucleotides are not externally labeled. For example, nucleotides can be naturally occurring nucleotides or synthetic analogs that do not include fluorescent moieties, dyes or other external optically detectable markers. Optionally, nucleotides do not include groups (such as dideoxy groups, reversible terminators and their analogs) that terminate nucleic acid synthesis. In other embodiments, nucleotides include marks or tags.

[0135] In some embodiments, the method for nucleic acid amplification comprises at least one cofactor, such as a cofactor that enhances the activity of DNA or RNA polymerase. In some embodiments, the cofactor comprises one or more divalent cations. Examples of divalent cations include magnesium, manganese and calcium. In various embodiments, the pre-implantation reaction mixture comprises a buffer containing one or more divalent cations. In illustrative embodiments, the buffer contains magnesium or manganese ions. In any embodiment of the present invention, the pre-implantation reaction or template reaction is started by adding a cofactor, especially a divalent cation. In some embodiments, the pre-implantation reaction mixture used for nucleic acid amplification herein may include at least one cofactor for recombinase assembly on nucleic acid or for homologous nucleic acid pairing. In some embodiments, the cofactor comprises any form of ATP, including ATP and ATPγS. In some embodiments, the method for nucleic acid amplification comprises at least one cofactor for regenerating ATP. For example, the cofactor may include an enzyme system that converts ADP into ATP. In some embodiments, the cofactor enzyme system is creatine phosphate and creatine kinase.

[0136] In any aspect of the present invention, the pre-implanted reaction mixture includes a component that partially denatures the template nucleic acid molecule. In some embodiments, partially denaturing conditions include treating the template nucleic acid molecule to be amplified with one or more enzymes or contacting the template nucleic acid molecule with the one or more enzymes, and the one or more enzymes can optionally partially denature the nucleic acid template in a sequence-specific or sequence-oriented manner (such as in an RPA reaction). In some embodiments, at least one enzyme optionally catalyzes chain invasion and / or unwinding in a sequence-specific manner. Optionally, one or more enzymes include one or more enzymes selected from the following: recombinases, topoisomerases, and helicases. In some embodiments, partially denaturing the template includes contacting the template with a recombinase and forming a nucleoprotein complex comprising a recombinase. Optionally, the template nucleic acid molecule is contacted with a recombinase in the presence of a first and optionally a second primer. Partial denaturation can include using a recombinase to catalyze chain exchange and hybridizing the first primer to the first primer binding sequence (or hybridizing the second primer to the second primer binding sequence). In some embodiments, partially denaturing comprises performing strand exchange using a recombinase, and hybridizing a first primer to a first primer binding sequence and hybridizing a second primer to a second primer binding sequence.

[0137] In some embodiments, partially denaturing the template nucleic acid molecule comprises contacting the template with one or more recombinases or nucleoprotein complexes. At least one of the nucleoprotein complexes may comprise a recombinase. At least one of the nucleoprotein complexes may comprise a primer (e.g., a first primer or a second primer, or a primer comprising a sequence complementary to a corresponding primer binding sequence in the template). In some embodiments, partially denaturing the template comprises contacting the template with a nucleoprotein complex comprising a primer. Partial denaturation may comprise hybridizing a primer of the nucleoprotein complex with a corresponding primer binding sequence in the template, thereby forming a primer-template double helix. In some embodiments, partially denaturing the template nucleic acid molecule comprises contacting the template with a first nucleoprotein complex comprising a first primer. Partial denaturation may comprise hybridizing a first primer of the first nucleoprotein complex with a first primer binding sequence of the forward strand, thereby forming a first primer-template double helix. In some embodiments, partially denaturing the template comprises contacting the template with a second nucleoprotein complex comprising a second primer. Partial denaturation may comprise hybridizing a second primer of the second nucleoprotein complex with a second primer binding sequence of the reverse strand, thereby forming a second primer-template double helix.

[0138] Therefore, the pre-implanted reaction mixture of the present disclosure and the templated reaction of the present disclosure include recombinase, and partial denaturation and / or amplification, including any one or more steps or methods described herein, can be achieved using recombinase and optionally recombinase auxiliary protein. The recombinase can include any agent capable of inducing a recombination event to occur or increasing its frequency of occurrence. The recombination event includes any event whereby two different polynucleotide chains are recombined with each other. Recombination may include homologous recombination. The recombinase may optionally be combined with (e.g., combined with) the first primer. In some embodiments, the enzyme catalyzing homologous recombination may form a nucleoprotein complex by binding to a single-stranded template nucleic acid molecule. In some embodiments, a homologous recombinase as a part of a nucleoprotein complex may bind to a homologous portion of a double-stranded polynucleotide. In some embodiments, the homologous portion of the polynucleotide hybridizes with at least a portion of the first primer. In some embodiments, the homologous portion of the polynucleotide is partially or completely complementary to at least a portion of the first primer. Suitable recombinases include RecA and its prokaryotic or eukaryotic homologs, or its functional fragments or variants, optionally in combination with one or more single-stranded binding proteins (SSB). In certain embodiments, the recombinase optionally coats the ssDNA to form nucleoprotein filaments that invade the double-stranded region of homology on the template.

[0139] In some embodiments, homologous recombinases catalyze strand invasion by forming a nucleoprotein complex and binding to homologous portions of a double-stranded polynucleotide to form a recombination intermediate with a three-strand structure (D-loop formation) (U.S. Pat. No. 5,223,414 to Zarling; U.S. Pat. Nos. 5,273,881 and 5,670,316 to Sena; and U.S. Pat. Nos. 7,270,981; 7,399,590; 7,435,561; 7,666,598; 7,763,427; 8,017,339; 8,030,000; 8,062,850; and 8,071,308, each of which is incorporated herein by reference in its entirety).

[0140] The recombinase of the reaction mixture, composition and kit comprises any suitable agent that can promote the recombination between polynucleotide molecules. The recombinase can be an enzyme that catalyzes homologous recombination. For example, the reaction mixture can comprise a recombinase that comprises or is derived from a bacterial, eukaryotic or viral (e.g., phage) recombinase.

[0141] In any one of the embodiments of the present invention, the homologous recombinase is a wild type, a mutant, a recombinant, a fusion or a fragment thereof. In some embodiments, the homologous recombinase comprises an enzyme from any organism, the organism comprising myoviridae (e.g., uvsX, RB69 and analogs thereof from bacteriophage T4), Escherichia coli (e.g., recA) or humans (e.g., RAD51). In an embodiment, the reaction mixture comprises one or more recombinases selected from the following: uvsX, RecA, RadA, RadB, Rad51, homologs thereof, functional analogs thereof, or combinations thereof. In illustrative embodiments, the recombinase is uvsX. The uvsX protein may be present, for example, at 50-1000 ng / μl, 100-750 ng / μl, 200-600 ng / μl or 250 to 500 ng / μl.

[0142] In some embodiments, the methods, kits and compositions for nucleic acid amplification include a pre-implanted reaction mixture containing one or more recombinase auxiliary proteins. For example, the auxiliary protein can increase the activity of the recombinase. In some embodiments, the auxiliary protein can bind to the single strand of the template nucleic acid molecule, or the recombinase can be loaded onto the template nucleic acid molecule. In some embodiments, the auxiliary protein is a wild type, a mutant, a recombinant, a fusion or a fragment thereof. In some embodiments, the auxiliary protein can be derived from any combination of the same or different species as the recombinase for performing the nucleic acid amplification reaction. The auxiliary protein can be derived from any bacteriophage, including myovirus phage. Examples of myoviral phages include T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, phage 31, phage 44RR2.8t, Rb49, phage Rb3, and phage LZ2. The accessory protein may be derived from any bacterial species, including Escherichia coli, Sulfolobus (e.g., S. solfataricus), or Methanococcus (e.g., M. jannaschii). In some embodiments, the method for nucleic acid amplification may comprise a single-stranded binding protein. The single-stranded binding protein comprises myovirus gp32 (eg, T4 or RB69), Sso SSB from Sulfolobus solfataricus, MjA SSB from Methanococcus jannaschii, and Escherichia coli SSB protein.

[0143] In certain embodiments, the method for nucleic acid amplification comprises the protein that improves the recombinase load to nucleic acid.For example, UvsY protein is a recombinase load protein.In certain embodiments, the reaction mixture comprises a recombinase auxiliary protein.In illustrative embodiments, the recombinase auxiliary protein is uvsY.UvsY can be between about 20 and 500ng / μl, for example, between about 20 and 250ng / μl or between about 20 and 125ng / μl.In non-limiting examples, UvsY is between 75 and 125ng / μl.

[0144] In any one embodiment of the present teachings, diffusion in a pre-implanted reaction mixture or a templated reaction mixture is limited by adding a diffusion-restricting agent, which effectively prevents or slows down the diffusion of one or more polynucleotide templates in a polynucleotide template and / or one or more amplification reaction products in an amplification reaction product through a pre-implanted or templated reaction mixture. When two or more template nucleic acid molecules are amplified in a single continuous liquid phase of a reaction mixture, it may be advantageous to include a diffusion-restricting agent. For example, a diffusion-restricting agent may prevent or slow down the diffusion of a template nucleic acid molecule or a polynucleotide produced by replication of at least a portion of a template nucleic acid molecule amplified in a pre-implanted or templated reaction mixture, thereby preventing the formation of polyclonal impurities without the need to compartmentalize the pre-implanted or templated reaction mixture by physical means or encapsulation means (e.g., emulsion) during amplification. Such methods of amplifying templates in a single continuous liquid phase of a single reaction mixture without compartmentalization greatly reduce the cost, time, and workload associated with the generation of libraries suitable for high-throughput methods (such as digital PCR, next-generation sequencing, and similar methods).

[0145] In certain embodiments, the diffusion-restricted agent is a screening agent. The screening agent can be any agent that effectively screens one or more template nucleic acid molecules or polynucleotides present in a pre-implantation or template reaction mixture, such as amplification reaction product and / or template nucleic acid molecules. In certain embodiments, the screening agent limits or slows down the migration of polynucleotide amplification products by pre-implantation or template reaction mixture. In certain embodiments, the average pore size of the screening agent makes the movement of the target component in the pre-implantation or template reaction mixture (e.g., polynucleotide) selectively delayed or prevented. In one example, the screening agent includes any compound that provides a matrix with multiple holes, and the multiple holes are small enough to slow down or delay the movement of polynucleotides or template nucleic acid molecules in the reaction mixture containing the screening agent. Therefore, the screening agent can reduce the Brownian motion of polynucleotides.

[0146] In some embodiments, the screening agent is a polymer compound. In some embodiments, the screening agent is a cross-linked or non-cross-linked polymer compound. By means of non-limiting examples, the screening agent may include polysaccharides, polypeptides, organic polymers or any other suitable polymers. In any one embodiment, the screening agent is a polymer in a straight or branched chain. In some embodiments, the screening agent is a charged or neutral polymer. In some embodiments, the screening agent may include a blend of one or more polymers each having a mean molecular weight and viscosity. In some embodiments, the screening agent is a polymer with a mean molecular weight between about 10,000 and 2,000,000Da: for example, between about 10,000 and 1,000,000Da, between about 10,000 and 500,000Da, between about 10,000 and 250,000Da or between about 10,000 and 100,000Da. In certain embodiments, the polymer has an average molecular weight between about 12,000 and 95,000 Da, or between about 13,000 and 95,000 Da.

[0147] In some embodiments, the sieving agent exhibits an average viscosity ranging from about 5 centipoise to about 15,000 centipoise when dissolved in water at 2 weight percent measured at about 25°C; or an average viscosity ranging from about 10 centipoise to about 10,000 centipoise when measured as a 2% aqueous solution at about 25°C; or an average viscosity ranging from about 15 centipoise to about 5,000 centipoise when measured as a 2% aqueous solution at about 25°C.

[0148] In some embodiments, the sieving agent has a molecular weight of about 25 to about 1,5000 kM. v , or about 75-1,000km v or about 85-800km v The viscosity average molecular weight (M v In some embodiments, the reaction mixture comprises the sieving agent at about 0.1 to about 20% weight / volume (w / v), or about 1-10% w / v, or about 2-5% w / v.

[0149] In certain embodiments, the screening agent is a polysaccharide polymer. In certain embodiments, the screening agent is a polymer of glucose or galactose. In certain embodiments, the screening agent is one or more of the following polymers: cellulose, dextran, starch, glycogen, agar, chitin, pectin or agarose. In certain embodiments, the screening agent is a pyranose glucopyranose polymer. In certain embodiments, the screening agent comprises a cellulose derivative, such as sodium carboxymethylcellulose, sodium carboxymethyl 2-hydroxyethylcellulose, methylcellulose, hydroxyethylcellulose, 2-hydroxypropylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethylcellulose, (hydroxypropyl)methylcellulose or hydroxyethylethylcellulose, or a mixture comprising any one or more of such polymers.

[0150] In some embodiments, the pre-implantation reaction mixture and / or the templated reaction mixture comprises a mixture of different sieving agents, such as a mixture of different cellulose derivatives, starches, polyacrylamides, and the like. In some embodiments, the pre-implantation reaction mixture comprises a crowding agent. In some embodiments, the pre-implantation reaction mixture comprises both a crowding agent and a sieving agent.

[0151] In some embodiments, the diffusion-reducing agent is a diffusion-reducing agent. Diffusion-reducing agents include any compound that reduces the migration of template nucleic acid molecules or polynucleotides from an area of ​​higher concentration to an area of ​​lower concentration. In some embodiments, diffusion-reducing agents include any compound that reduces the migration of any component of a nucleic acid amplification reaction regardless of size.

[0152] In some embodiments, the diffusion-restricted agent is a drug compound. The term "drug compound" and its variants refer to any composition, such as a chemical composition, that can be connected to a nucleic acid and prevent it from diffusing through a reaction mixture, but still allows the use of such polynucleotides, primers, templates or amplification products in a nucleic acid synthesis reaction for nucleic acid synthesis. The connection of such drug compounds to nucleic acids in a synthesis reaction usually reduces the mobility of such nucleic acids in a reaction mixture, and can be used to prevent cross-contamination of amplification products or templates between different synthesis reactions occurring in the same reaction mixture. In some embodiments, nucleic acids are modified to be connected or combined with drug compounds. For example, an affinity portion, such as a linker portion, can be connected to nucleic acids and the drug compound is a binding partner portion, such as a receptor type portion, which is combined with an affinity portion. In some embodiments, nucleic acids are connected to a biotin portion, which can be combined with an avidin-like portion used as a drug compound. The avidin-like portion includes avidin and any derivatives, analogs, and other non-natural forms of avidin (e.g., neutravidin) of avidin bound to biotin. In some embodiments, the connection of a drug component to one or more nucleic acid components can increase the number or proportion of monoclonal products. For example, by changing the overall size, length, radius, shape or charge of the modified nucleic acid compared to the nucleic acid lacking the attached compound, the drug compound can provide hydrodynamic resistance when attached to the nucleic acid. In some embodiments, the drug compound attached to the nucleic acid can change the interaction between the nucleic acid and the aqueous medium compared to the interaction between the aqueous medium and the nucleic acid lacking the attached compound. In some embodiments, the drug compound can be synthetic, recombinant or naturally occurring. In some embodiments, the drug compound can be charged, uncharged, polar or hydrophobic. In some embodiments, the drug compound can be linear, branched or have a dendritic structure. In some embodiments, the drug compound can include a single part or polymer of a nucleoside, a carbohydrate, a lipid or an amino acid. Optionally, the drug compound includes a sugar moiety, a polysaccharide, a protein, a glycoprotein or a polypeptide. Optionally, the drug compound includes BSA, lysozyme, β-actin, myosin, lactalbumin, ovalbumin, β-galactosidase, lactate dehydrogenase or an immunoglobulin (e.g., IgG). Optionally, the pharmaceutical compound that alters the mobility of nucleic acids through aqueous media includes one or more polyethylene oxide (PEO) or polypropylene oxide (PPO) moieties, polymers comprising polyethylene oxide (PEO) or polypropylene oxide (PPO). Non-limiting examples of such polymers include triblock copolymers (e.g., PEO-PPO-PEO), Pluronics TMType polymers and hydrophobically modified PEO polymers. Optionally, the drug compound includes one or more amino acid moieties, polypeptides and polypeptide-like compounds. Optionally, the drug compound includes a sugar moiety, a polysaccharide, a hydrophobically modified polysaccharide, a cellulose derivative, sodium carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose or hydroxypropylmethylcellulose. Optionally, the drug compound includes a hydrophobically modified alkali-soluble associative (HASE) polymer, a hydrophobically modified polyacrylamide, a thermally responsive polymer or N-isopropylacrylamide (NTPAAm), and optionally, the drug compound includes poly (ethylene glycol) methyl ether acrylate (PEGMEA), tetraethylene glycol diacrylate (TEGDA), poly (ethylene glycol) dimethacrylate (EGDMA) or N, N'-methylene-bis-acrylamide (NMBA). Optionally, the drug compound includes a protein or polypeptide, including BSA, lysozyme, β-actin, myosin, lactalbumin, ovalbumin, β-galactosidase or lactate dehydrogenase.

[0153] It should be noted that the concepts of sieving agents and diffusion reducers are not necessarily mutually exclusive; sieving agents can often effectively reduce the diffusion of target compounds in the reaction mixture, while diffusion reducers can often have a sieving effect on the reaction components. In some embodiments, the same compound or pre-implanted reaction mixture additive can be used as a sieving agent and / or a diffusion reducer. In some embodiments, any sieving agent of the present invention can be capable of acting as a diffusion reducer, and vice versa.

[0154] In some embodiments, the diffusion reducing agent and / or sieving agent comprises polyacrylamide, agar, agarose, or a cellulose polymer, such as hydroxyethyl cellulose (HEC), methyl-cellulose (MC), or carboxymethyl cellulose (CMC).

[0155] In some embodiments, the pre-implantation reaction mixture contains a sieving agent and / or diffusion reducing agent at a concentration of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90% or 95% w / v (weight of agent / unit volume of reaction mixture).

[0156] In some embodiments, the diffusion-limiting agent is a crowding agent. For example, a crowding agent can increase the concentration of one or more components in a nucleic acid amplification reaction by creating a crowded reaction environment. In some embodiments, the pre-implanted and / or templated reaction mixture comprises a sieving agent and / or a diffusion agent as well as a crowding agent.

[0157] In some embodiments, different template nucleic acid molecules are pre-implanted into one or more different discrete carriers (e.g., beads or particles) without the need for compartmentalization before amplification. In other embodiments, before amplification, template nucleic acid molecules are distributed (partitioned / distributed) into an emulsion. Contrary to amplification in a single continuous liquid phase, pre-implantation reactions can be performed in parallel in multiple partitioned reaction volumes. Each reaction volume can include a pre-implantation reaction mixture. For example, template nucleic acid molecules can be distributed or placed in a reaction chamber array or reaction volume array so that at least two such compartments or volumes in the array accept a single template nucleic acid molecule. In some embodiments, multiple individual reaction volumes are formed. Before amplification, the reaction chamber (or reaction volume) can be optionally sealed. Pre-implantation reactions can be performed in each reaction chamber to produce a substantially monoclonal template nucleic acid molecule population. In another embodiment, the reaction mixture is partitioned or partitioned into a plurality of microreactors dispersed in the continuous phase of the emulsion. Each compartment or microreactor serves as an independent amplification reactor, so that the entire emulsion can support many individual amplification reactions in a single (non-continuous) liquid phase in a single reaction vessel (e.g., Eppendorf tube or hole). As used herein, the term "emulsion" includes any composition comprising a mixture of a first liquid and a second liquid, wherein the first and second liquids are substantially immiscible with each other. The partitioned or separate reaction volumes are optionally not mixed or connected with each other, or are not capable of mixing or connecting with each other. The pre-implantation reaction mixture in the microreactor can be any pre-implantation reaction mixture discussed herein.

[0158] In some embodiments, the method of nucleic acid synthesis further comprises recovering at least some of the vectors attached to the substantially monoclonal population of template nucleic acid molecules from the emulsion. In some embodiments, the method of nucleic acid synthesis further comprises placing at least some of the vectors attached to the substantially monoclonal population of template nucleic acid molecules on a surface. In some embodiments, the method of nucleic acid synthesis further comprises forming an array by placing at least some of the vectors attached to the substantially monoclonal population of template nucleic acid molecules on a surface.

[0159] In some embodiments, the present disclosure generally relates to compositions, and systems, methods, kits, and devices, which include pre-implanted reaction mixtures and templated reaction mixtures. Thus, in certain embodiments, provided herein are reaction mixtures comprising a polymerase and one or more pre-implanted vectors. The reaction mixture composition may include a recombinase and optionally a recombinase auxiliary protein as known for RPA reactions. In such embodiments, the reaction mixture, particularly the pre-implanted reaction mixture, may include 5×10 7 with 10 9 template nucleic acid molecules, the solution contains 5×10 6 with 10 10In some examples, the templated reaction mixture is contained in less than 1,000,000, 500,000, 1,000, 500, 100, 10 or 0 template nucleic acid molecules in the solution, and the pre-implanted solid carrier of the present invention. In the pre-implanted and templated reaction mixture, polymerase and optionally recombinase are usually present in amplification effective concentration, as known to RPA reaction, or exist in higher concentration, so that it can be merged into the final pre-implanted reaction mixture with other reaction components. In any embodiment of the present teachings, the volume of the preimplantation reaction mixture and / or the templated reaction mixture is between about 50 and 2,000 μl, e.g., between about 50 and 1,500 μl, between about 50 and 1,000 μl, between about 50 and 500 μl, between about 50 and 250 μl, or between about 50 and 150 μl. In any disclosed embodiment, the volume of the preimplantation reaction mixture is different from the volume of the templated reaction mixture.

[0160] The pre-implanted reaction mixture and the templated reaction mixture may further comprise other components. For example, the composition may comprise nucleotides, a first primer population, optionally a second primer, a cofactor, and a buffer. The first primer population and optionally the second primer population may be connected to one or more carriers. As a non-limiting example, the composition comprises: one or more carriers; a recombinase, such as uvsX; a polymerase, such as Sau DNA polymerase; a recombinase load protein, such as uvsY; a single-stranded binding protein, such as gp32 protein; nucleotides; ATP; creatine phosphate; and creatine kinase. The composition may be in liquid form, or it may be in solid form, such as a dried-down pellet form that can be rehydrated. In addition, the components of the composition may be separated so that any combination of components may be in the form of a precipitate or a liquid, and one or more combinations of the remaining components may be in the form of one or more separate precipitates or liquids. Such combinations may form a kit comprising at least two of such combinations. For example, a kit may comprise a precipitate of all reaction mixture components of the teachings of the present invention except for a polymerase, which may be provided in the kit as a separate precipitate or liquid.

[0161] In illustrative embodiments, the composition comprises a population of template nucleic acid molecules, a polymerase, a recombinase, a forward primer, a reverse primer, nucleotides, and a buffer. In some embodiments, the composition comprises a template nucleic acid molecule, a forward primer, a reverse primer, a uvsX recombinase, a uvsY recombinase load protein, a gp32 protein, a Sau DNA polymerase, dNTPs, ATP, creatine phosphate, and creatine kinase.

[0162] In some embodiments, the composition comprises at least two different template nucleic acid molecules with a first primer binding sequence and a second primer binding sequence, a recombinase, a recombinase auxiliary protein, a polymerase, a first universal primer, a second universal primer, dNTPs and a buffer. In some embodiments, the composition further comprises one or more carriers. In an illustrative embodiment, the composition comprises at least two different template nucleic acid molecules with a first primer binding sequence and a second primer binding sequence, a uvsX recombinase, a uvsY recombinase load protein, a gp32 protein, a Sau DNA polymerase, ATP, creatine phosphate, creatine kinase, a first universal primer connected to a bead carrier, a second universal primer and a buffer.

[0163] In some embodiments, by adding each component separately to the aqueous solution or emulsion solution, a pre-implantation reaction mixture or a templated reaction mixture is formed. In other embodiments, the reaction mixture is in the form of a dehydrated precipitate that needs to be rehydrated before use. The dehydrated precipitate can include, for example, a recombinase, an optional recombinase auxiliary protein, optionally gp32, a DNA polymerase, dNTPs, ATP, optionally creatine phosphate, optionally a crowding agent, and optionally creatine kinase. The rehydration buffer can include, for example, Tris buffer, potassium acetate, and optionally a crowding agent such as PEG. The DNA polymerase can be, for example, T4 or T7 DNA polymerase, and can further include thioredoxin when the polymerase is T7 DNA polymerase. In some embodiments, when using a dehydrated precipitate comprising a reaction mixture component, the precipitate is rehydrated with a rehydration buffer, and the template nucleic acid molecule, primer, and additional nuclease-free water are added to the final volume.

[0164] In some embodiments, the pre-implanted reaction mixture or the templated reaction mixture is pre-incubated under conditions that inhibit the start of premature reactions. For example, one or more components in the pre-implanted reaction mixture may be withheld from the reaction vessel to prevent the start of premature reactions. In order to start the reaction, a divalent cation (e.g., magnesium or manganese) is added. In another example, the pre-implanted reaction mixture is pre-incubated at a temperature that inhibits enzyme activity. The reaction may be pre-incubated at about 0-15°C or about 15-25°C to inhibit the start of premature reactions. Subsequently, the reaction is incubated at a higher temperature to increase enzymatic activity. In an illustrative embodiment, the pre-implanted reaction mixture and / or the templated reaction mixture is not exposed to a temperature above 42°C during the reaction.

[0165] In some embodiments of the disclosed embodiments, the pre-implantation and / or templated reaction optionally comprises repeated cycles of nucleic acid amplification. In some embodiments, the amplification cycle comprises (a) partial, incomplete or complete denaturation or dissociation of a chain of a double-stranded nucleic acid, (b) hybridization or annealing of a primer with a partially or completely single-stranded nucleic acid, and (c) primer extension to form an extended primer chain. In some embodiments, the amplification cycle optionally comprises: (a) hybridization of a first primer with a template chain, (b) primer extension to form a first extended chain, (c) partial or incomplete denaturation of an extended chain from a template chain. Optionally, the denatured portion of the template chain from step (c) is arbitrarily hybridized with a different first primer in the next amplification cycle. In some embodiments, primer extension in an amplification cycle involves displacing a chain of a duplex nucleic acid from another chain of the duplex or displacing a first extended chain from a template chain. A second primer that hybridizes to the 3′ end of the first extended chain may be included. In some embodiments, the disclosed methods (and related compositions, systems, and kits) further comprise one or more primer extension steps. For example, the method comprises extending a primer by incorporating nucleotides using a polymerase. In an embodiment, the primer extension is included in contacting the hybridized primer with a polymerase and one or more types of nucleotides under nucleotide incorporation conditions. Typically, the primer extension occurs in a template-dependent manner. Optionally, the disclosed method (and related compositions, systems, and kits) includes extending the first primer by using a polymerase, incorporating one or more nucleotides into the 3'OH of the first primer of the first primer-template double helix, thereby forming an extended first primer. Optionally, the disclosed method (and related compositions, systems, and kits) includes binding the second primer binding sequence of the first extended primer to the second primer by any suitable method (such as joining or hybridization). Optionally, the disclosed method (and related compositions, systems, and kits) includes extending the second primer by using a polymerase, incorporating one or more nucleotides into the second primer of the second primer-template double helix, thereby forming an extended second primer.

[0166] In some embodiments, the first primer is extended so as to form a first extended primer. The first extended primer may include some or all of the template reverse strand sequence. Optionally, the first extended primer includes a second primer binding sequence. In some embodiments, the second primer is extended so as to form a second extended primer. The second extended primer may include some or all of the template forward strand sequence. Optionally, the second extended primer includes a first primer binding sequence. In some embodiments, the method (and related compositions, systems, and kits) may further include connecting one or more extended primer chains to a carrier. Optionally, connection may be performed during amplification or alternatively after amplification is completed. In some embodiments, the carrier is connected to a first primer population. For example, the carrier may include a first primer population, and the method may include hybridizing at least one of the extended second primers with the first primer of the carrier, thereby connecting the extended second primer to the carrier. For example, the first primer may hybridize with the first primer binding sequence in the extended second primer. In some embodiments, the carrier includes multiple instances of the second primer, and the method includes hybridizing at least one of the extended first primer chains with the second primer of the carrier, such as in bridge PCR.

[0167] In any one embodiment of the teachings of the present invention, a pre-implantation reaction is performed using an RPA reaction, wherein a polymerase, a recombinase, and usually a recombinase auxiliary protein are used to achieve partial denaturation and / or amplification, comprising any one or more steps or methods described herein. Without being limited by theory, it is believed that the recombinase coats single-stranded DNA (ssDNA) to form nucleoprotein filaments that invade homologous double-stranded regions on the template nucleic acid molecule. This produces short hybrids and displacement chain bubbles known as D-loops. Using DNA polymerase, the free 3' end of the hybridization primer is extended to synthesize a new complementary chain. The complementary chain displaces the initially paired partner chain of the template nucleic acid molecule as it is elongated. In one embodiment, one or more of a pair of primers is contacted with one or more recombinases before contacting the template nucleic acid molecule (which is optionally double-stranded). The RPA reaction is typically an isothermal reaction and is performed in an emulsion.

[0168] In any embodiment of the present teachings, a pre-implantation reaction is performed by template walking, wherein portions of a double-stranded nucleic acid molecule become dissociated, allowing a primer to bind to one strand to initiate a new round of replication (see, e.g., U.S. Patent Publication No. 2012 / 0156728, published on June 21, 2012, which is incorporated herein by reference in its entirety). An embodiment of template walking includes a primer extension method comprising: (a) a primer hybridization step, (b) an extension step, and (c) a walking step. Optionally, the primer hybridization step comprises hybridizing a first primer to a first primer binding sequence on a template nucleic acid molecule ("reverse strand"). Optionally, the extension step comprises generating an extended first forward strand, the extended first forward strand being a full-length complementary sequence of the reverse strand and hybridizing therewith. For example, the extended first forward strand is generated by extending the first forward primer in a template-dependent manner using the reverse strand as a template. Optionally, the walking step comprises hybridizing another first primer to the first primer binding sequence, wherein the reverse strand also hybridizes to the first forward strand. For example, the walking step comprises denaturing at least a portion of the first primer binding sequence from the forward strand, wherein another portion of the reverse strand is still hybridized to the forward strand. In any disclosed embodiment, the reaction mixture comprises one or more carriers having a first primer bound thereto, wherein the first primer binding sequence on at least one of the template nucleic acid molecules is complementary or identical to at least a portion of the first primer. In some embodiments, at least one of the template nucleic acid molecules has a second primer binding sequence that is complementary or identical to at least a portion of the second primer. In some embodiments, the second primer is also bound to the carrier so that amplification can occur back and forth on the surface. In various embodiments, the second primer is in solution. In other embodiments, the second primer is fixed to a carrier. The template walking reaction is typically performed isothermally and in an emulsion.

[0169] Template walking can introduce many identical nucleotides at the proximal end of the template nucleic acid molecule connected to the carrier. In some embodiments, template walking is performed on one or more carriers in a manner such that the connected template nucleic acid molecule introduces less than 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250 or 300 identical nucleotides at the proximal end of the template nucleic acid molecule connected to the carrier. In other embodiments, template walking is performed on one or more carriers in a manner such that the connected template nucleic acid molecule introduces about 10 and 300 identical nucleotides at the proximal end of the template nucleic acid molecule connected to the carrier, for example, about 10 and 200 identical nucleotides, about 10 and 150 identical nucleotides, or about 10 and 100 identical nucleotides at the proximal end of the template nucleic acid molecule connected to the carrier. In any embodiment, the proximal end is the end of the template nucleic acid molecule that is closest to the carrier to which each template nucleic acid molecule is connected. In some aspects, the template nucleic acid molecule connected to the carrier is a series of identical nucleotides connected to the template nucleic acid molecule or the template nucleic acid segment. In some embodiments, less than 5%, 10%, 15%, 20% or 25% of each of the substantially monoclonal template nucleic acid molecule population has a variable number of identical nucleotides at the proximal end. For example, less than 5%, 10%, 15%, 20% or 25% of each of the substantially monoclonal template nucleic acid molecule population can introduce between about 10 and 300 identical nucleotides at the proximal end of the template nucleic acid molecule connected to the carrier, such as between about 10 and 200 identical nucleotides, between about 10 and 150, between about 10 and 100, or between about 20 and 50 identical nucleotides at the proximal end of the template nucleic acid molecule connected to the carrier.

[0170] In some embodiments of the disclosed embodiments, a PCR method is used to perform the pre-implantation reaction. Those skilled in the art will recognize various methods for performing PCR that will produce a substantially monoclonal template nucleic acid molecule population. In some embodiments, the pre-implantation reaction is performed in a single round or single cycle PCR. In other embodiments, the pre-implantation reaction is performed in multiple rounds or multi-cycle PCR. For example, in some pre-implantation methods using amplification (e.g., PCR) cycles, one or two or more PCR cycles can be performed in the absence of a vector to produce the desired template and / or its amount, which can be followed by implanting the desired template nucleic acid onto the vector by one, or two or more cycles of PCR in the presence of the vector (see, e.g., Figure 7). For example, the method may comprise diluting a certain amount of template nucleic acid molecules reacted with the vector to reduce the percentage of vectors reacted with more than one template nucleic acid molecule. In some embodiments, the template nucleic acid molecules are diluted so that the pre-implantation reaction has a vector: template nucleic acid molecule ratio, and the ratio is selected to optimize the percentage of vectors having a substantially monoclonal population of template nucleic acid molecules connected thereto. For example, the pre-implantation reaction can be performed with the following vector: template nucleic acid molecule ratio: at least about 1: 1, 1.25: 1, 1.5: 1, 1.75: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 50: 1, 75: 1, and 100: 1. In some embodiments, PCR is performed in batches in a solution of a single reaction mixture. In some embodiments, PCR is performed in a well or reaction chamber in a continuous solution. In some embodiments, PCR is performed in an emulsion, wherein PCR is performed in a plurality of microreactors in the emulsion as described elsewhere herein.

[0171] The described method for pre-implantation carrier is included in fixing one or more reaction components (such as one or more template nucleic acid molecules and / or primers) during the pre-implantation reaction, to prevent the cross contamination of amplification reaction product and the consequent monoclonality reduction. Such example comprises bridge amplification, wherein all primers (such as forward and reverse primers) required for amplification are connected to the surface of the matrix carrier. Except such fixing, other fixed components are included in the reaction mixture. For example, template nucleic acid molecules and / or amplification primers can be suspended in gel or other matrix during amplification, to prevent the amplification reaction product from migrating from the synthesis site. Such gel and matrix usually need to be removed afterwards, thus need to use appropriate " melting " or other recovery steps and consequent yield loss.

[0172] In any one embodiment of the present teachings, the pre-implantation and / or templated reaction is performed under isothermal conditions. In some embodiments, the isothermal conditions include subjecting the reaction to a temperature change constrained within a limited range during at least some partial amplification (or the entire amplification process), including, for example, a temperature change equal to or less than about 10°C, or about 5°C, or about 1-5°C, or about 0.1-1°C or less than about 0.1°C, or, for example, a temperature change equal to or less than 10°C, or 5°C, or 1-5°C, or 0.1-1°C or less than 0.1°C. The temperature of the isothermal reaction may typically be between about 15°C and 65°C, for example, between about 15°C and 55°C, between about 15°C and 45°C, between about 15°C and 37°C, between about 30°C and 60°C, between about 40°C and 60°C, between about 55°C and 60°C, between about 35°C and 45°C, or between about 37°C and 42°C. In other embodiments, the pre-implantation reaction is not exposed to temperatures above 40°C, 41°C, 42°C, 43°C, 45°C or 50°C. Thus, in certain embodiments, the reaction mixture is not exposed to hot start conditions. However, it should be understood that the enzymes used at these temperatures will require combinatorial optimization, and may require changes in enzymes, such as using different DNA polymerases, such as Bst instead of Bsu. The rate limiting enzyme can be a polymerase, wherein a high concentration or excess (i.e., non-restrictive) amount of polymerase or a lower temperature ensures that the amplification reaction is carried out based on the kinetics of the polymerase.

[0173] The pre-implantation and / or templated reaction can be performed for 0.25 minutes to 240 minutes, thereby amplifying the nucleic acid template. In certain embodiments, the pre-implantation reaction is performed for about 0.25 to 240 minutes, such as about 0.25 to 120 minutes, about 0.25 to 60 minutes, about 0.25 to 30 minutes, about 0.25 to 15 minutes, about 0.25 to 10 minutes, about 0.25 to 7.5 minutes, about 0.25 to 5 minutes or about 2 to 5 minutes. In further illustrative embodiments, the pre-implantation and / or templating reaction is performed for about 1.5 to 20 minutes, such as about 1.5 to 15 minutes, about 1.5 to 10 minutes, about 1.5 to 8 minutes, about 1.5 to 6 minutes, about 1.5 to 5 minutes, or about 1.5 to 4 minutes, and optionally, the reaction is isothermal and the temperature of the reaction is between about 35° C. and 65° C., such as between about 35° C. and 55° C., between about 35° C. and 45° C., between about 30° C. and 60° C., between about 40° C. and 60° C., between about 40° C. and 55° C., between about 50° C. and 60° C., or between about 37° C. and 42° C. For example, the pre-implantation reaction can be performed for about 1 to 10 minutes in an isothermal reaction, wherein the reaction temperature can be between about 35° C. and 45° C., or the pre-implantation reaction can be performed for about 2 to 5 minutes in an isothermal reaction, wherein the reaction temperature can be between about 37° C. and 42° C. In some embodiments, the pre-implantation reaction is performed using one or more PCR amplification cycles and the total reaction time can be between about 1.5 and 50 minutes, between about 1.5 and 45 minutes, between about 1.5 and 40 minutes, between about 1.5 and 35 minutes, between about 1.5 and 30 minutes, between about 1.5 and 25 minutes, between about 1.5 and 20 minutes, between about 1.5 and 15 minutes, between about 1.5 and 14 minutes, between about 1.5 and 13 minutes, between about 1.5 and 12 minutes, between about 1.5 and 10 minutes, between about 1.5 and 8 minutes, between about 1.5 and 6 minutes, between about 1.5 and 5 minutes, or between about 1.5 and 4 minutes.

[0174] In some embodiments, the method is performed without subjecting the double-stranded template nucleic acid molecule to extreme denaturing conditions during amplification. For example, the method can be performed without subjecting the template nucleic acid molecule to a T of equal to or greater than the template during amplification. m In some embodiments, the method is performed without contacting the template with a chemical denaturant (such as NaOH, urea, guanidine salts, and the like) during amplification. In some embodiments, amplification comprises isothermal amplification.

[0175] In some embodiments, the method is performed without subjecting the template nucleic acid molecule to extreme denaturing conditions during between about 2 and 50 consecutive cycles, between about 2 and 40 consecutive cycles, between about 2 and 30 consecutive cycles, between about 2 and 25 consecutive cycles, between about 2 and 20 consecutive cycles, or between about 2 and 15 consecutive cycles. For example, the method can include between about 2 and 50 consecutive cycles, between about 2 and 40 consecutive cycles, between about 2 and 30 consecutive cycles, between about 2 and 25 consecutive cycles, between about 2 and 20 consecutive cycles, or between about 2 and 15 consecutive nucleic acid synthesis cycles without contacting the nucleic acid template with a chemical denaturant or raising the temperature above 50°C or 55°C. In some embodiments, the method comprises performing between about 2 and 50 consecutive cycles, between about 2 and 40 consecutive cycles, between about 2 and 30 consecutive cycles, between about 2 and 25 consecutive cycles, between about 2 and 20 consecutive cycles, or between about 2 and 15 consecutive cycles of nucleic acid synthesis without subjecting the nucleic acid template to an actual or calculated T greater than that of the template or population of templates. m (or actual or calculated mean T for a template or template population) m ) a temperature lower than 25°C, 20°C, 15°C, 10°C, 5°C, 2°C or 1°C. Continuous nucleic acid synthesis cycles may or may not include intervening partial denaturation and / or primer extension steps. Optionally, at least one cycle of template-based replication includes a partial denaturation step, an annealing step and an extension step. In some embodiments, the connected identical primers in the pre-implantation reaction are a sequence of adenosine or uridine of between about 5 and 100 nucleotides in length or a certain combination of adenosine and uridine, such as a length of between about 5 and 80 nucleotides, a length of between about 5 and 60 nucleotides, a length of between about 5 and 40 nucleotides, a length of between about 10 and 75 nucleotides, a length of between about 15 and 75 nucleotides, or a length of between about 20 and 50 nucleotides.

[0176] In some embodiments, the pre-implantation and / or templated reaction results in the formation of a pre-implanted and / or templated vector population having a substantially monoclonal template nucleic acid molecule population attached thereto. In various aspects, a substantially monoclonal template nucleic acid molecule population is a template nucleic acid molecule having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity at the sequence level. In some embodiments, the percentage of substantially monoclonal template nucleic acid molecules attached to the pre-implanted and / or templated vector is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the template nucleic acid molecules attached thereto.

[0177] In various embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the vectors in the preimplanted and / or templated vector population have a substantially monoclonal population of nucleic acid molecules attached during the preimplantation and / or templated reaction. In illustrative embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the vectors in the preimplanted and / or templated vector population have a substantially monoclonal population of nucleic acid molecules attached during the preimplantation and / or templated reaction.

[0178] In some embodiments, the pre-implantation reaction can produce: a carrier (empty solid carrier) to which zero template nucleic acid molecules are connected, other pre-implanted carriers to which one type of template nucleic acid molecules are connected, and other pre-implanted carriers to which more than one type of template nucleic acid molecules are connected. In any embodiment of the present invention, the number of connected template nucleic acid molecules in a population of substantially monoclonal template nucleic acid molecules connected to one or more pre-implanted carriers is a pre-implantation number. In any embodiment of the disclosed embodiments, the pre-implantation number is 1 or about 1 to 150,000 template nucleic acid molecules, such as about 1 to 100,000, about 1 to 75,000, about 1 to 50,000, about 1 to 25,000, about 1 to 10,000, about 1 to 5,000, or about 1 to 2,500 template nucleic acid molecules. In illustrative embodiments, the pre-implanted number is between about 10 and 100,000 template nucleic acid molecules, such as between about 10 and 75,000, between about 10 and 50,000, between about 10 and 25,000, between about 10 and 10,000, between about 10 and 5,000, or between about 10 and 2,500 template nucleic acid molecules.

[0179] In some embodiments, after the pre-implantation reaction, most of any primers connected to the carrier do not bind to the template nucleic acid molecule. These unbound primers can be used in subsequent templated reactions for further amplification of the template nucleic acid molecule. For example, after the pre-implantation reaction, at least 90%, 95%, 96%, 97%, 98% or 99% of the primers connected to the carrier are generally not bound to the template nucleic acid molecule, or all primers except one of the primers connected to the carrier are not bound to the template nucleic acid molecule.

[0180] In some embodiments, the present disclosure generally relates to methods, and systems, compositions, kits and devices, wherein the methods are generally included in a templated reaction after a pre-implantation reaction, wherein the template nucleic acid molecules on the pre-implanted carrier are further amplified (referred to herein as a templated reaction). The templated reaction mixture does not include additional template nucleic acid molecules in the solution, so that before the templated reaction is started, the template nucleic acid molecules connected to one or more pre-implanted carriers are the main source or sole source of template nucleic acid molecules in the templated reaction mixture. In illustrative embodiments, one or more washes are performed on one or more pre-implanted carriers before being introduced into the templated reaction mixture. In some embodiments, at the end of the pre-implantation reaction mixture, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1% of the template nucleic acid molecules in the solution are present in the templated reaction. In any embodiment of the present teachings, the percentage of template nucleic acid molecules in the templated reaction mixture that are attached (pre-implanted) to one or more carriers prior to initiating the templated reaction is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the template nucleic acid molecules in the reaction mixture.

[0181] In any one embodiment of the present teachings, the templated reaction is typically an RPA reaction. Therefore, any details about the components and conditions of the RPA reaction discussed above for the pre-implantation reaction are applicable to the templated reaction, except that the template nucleic acid is typically present in the initial reaction mixture of the pre-implantation reaction, but not in the initial reaction mixture of the templated reaction. The templated reaction mixture typically includes all or some of the following: one or more pre-implanted solid supports, which include a substantially identical first primer population connected, and have a substantially monoclonal template nucleic acid molecule connected thereto, such as one template molecule; polymerase; recombinase; optional single-stranded binding protein; optional recombinase load protein; optional second primer or reverse primer, which can be connected to the solid support, but in the illustrative example it is in solution; dNTP; ATP; buffer; and optionally one or two of phosphocreatine and creatine kinase. Divalent cations, such as MgCl2 or Mg(OAc)2, may be added to start the reaction. In various embodiments, the buffer includes a crowding agent, such as PEG; Tris buffer; and / or potassium acetate. The forward primer binding sequence on the template nucleic acid molecule is complementary to or identical to at least a portion of the forward primer, and the optional reverse primer binding sequence on the template nucleic acid molecule is complementary to or identical to at least a portion of the reverse primer. The reaction mixture of the templated reaction itself forms an individual aspect of the present invention. In other illustrative embodiments, the RPA templated reaction is a large batch isothermal amplification, or is performed in the wells of a porous solid support (e.g., see the pre-implanted amplification reaction discussed further herein).

[0182] In some embodiments, the templated reaction mixture is pre-incubated under conditions that inhibit the start of premature reactions. For example, one or more components in the templated reaction mixture may be withheld from the reaction vessel to prevent premature reactions from starting. In order to start the reaction, a divalent cation (e.g., magnesium or manganese) is added. In another example, the templated reaction mixture is pre-incubated at a temperature that inhibits enzyme activity. The reaction is pre-incubated at about 0-15°C or about 15-25°C to inhibit the start of premature reactions. Subsequently, the reaction may be incubated at a higher temperature to increase enzymatic activity. In an illustrative embodiment, the templated reaction mixture is not exposed to a temperature above 42°C during the reaction.

[0183] Since the templated reaction is usually an RPA reaction, it is carried out under isothermal conditions. In some embodiments, the isothermal conditions include subjecting the reaction to a temperature change constrained within a limited range during at least some partial amplification (or the entire amplification process), including, for example, a temperature change equal to or less than about 10°C, or about 5°C, or about 1-5°C, or about 0.1-1°C or less than about 0.1°C, or, for example, a temperature change equal to or less than or 10°C, or 5°C, or 1-5°C, or 0.1-1°C or less than 0.1°C. The temperature of the isothermal reaction may be typically between about 15°C and 65°C, for example, between about 15°C and 55°C, between about 15°C and 45°C, between about 15°C and 37°C, between about 30°C and 60°C, between about 40°C and 60°C, between about 55°C and 60°C, between about 35°C and 45°C, or between about 37°C and 42°C about 15°C. In other embodiments, the pre-implantation reaction is not exposed to temperatures above 40°C, 41°C, 42°C, 43°C, 45°C or 50°C. Thus, in certain embodiments, the reaction mixture is not exposed to hot start conditions. However, it should be understood that the enzymes used at these temperatures will require combinatorial optimization, and may require changes in enzymes, such as using different DNA polymerases, such as Bst instead of Bsu. The rate limiting enzyme can be a polymerase, wherein a high concentration or excess (i.e., non-restrictive) amount of polymerase or a lower temperature ensures that the amplification reaction is carried out based on the kinetics of the polymerase.

[0184] In any one embodiment of the present teachings, the templated reaction is typically an RPA reaction, which is carried out for about 0.25 to 240 minutes, such as about 0.25 to 120 minutes, about 0.25 to 60 minutes, about 0.25 to 30 minutes, about 0.25 to 15 minutes, about 0.25 to 10 minutes, about 0.25 to 7.5 minutes, about 0.25 to 5 minutes or about 2 to 5 minutes. In an illustrative embodiment, the templated reaction is carried out for about 10 to 120 minutes, such as about 10 to 60 minutes, about 10 to 45 minutes, about 10 to 30 minutes or about 10 to 20 minutes. In other illustrative embodiments, the templated reaction is carried out for about 20 to 60 minutes, such as about 20 to 50 minutes, about 20 to 40 minutes, about 20 to 35 minutes or about 20 to 30 minutes. In some embodiments, one or more templated reactions are carried out (or the templated reaction is carried out in two steps of two separate amplifications, such as two separate RPA reactions). In such embodiments, separate templated or RPA reactions can be performed for the same or different amounts of time. For example, in a two-step templated method comprising two separate isothermal amplification reactions (e.g., RPA reactions), the first reaction can be performed for about 2.5 minutes and the second or subsequent reaction can be performed for about 20 minutes.

[0185] In any embodiment of the present teachings, the templated reaction comprises amplifying a population of different template nucleic acid molecules on one or more pre-implanted vectors to produce one or more templated vectors. For example, after one or more templated reactions, the ligated substantially monoclonal template nucleic acid molecules present on the templated vectors can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 25,000, 50,000, 100,000, 250,000, 500,000, or 10 6 In some embodiments, after one or more template reactions, there may be about 100,000 to 10 6 In some embodiments, at least 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 25,000, or 50,000 times as many linked substantially monoclonal template nucleic acid molecules are present on the templated vector as on the pre-implanted vector. In some embodiments, only about 1 or only 1 template nucleic acid molecule is present on each pre-implanted vector. In some embodiments, at least 50,000, 75,000, or 100,000 substantially monoclonal template nucleic acid molecules or between about 25,000 and 1,000,000 substantially monoclonal template nucleic acid molecules are present on the pre-implanted solid support, such as between about 25,000 and 500,000, between about 25,000 and 250,000, between about 25,000 and 125,000, or between about 25,000 and 100,000 substantially monoclonal template nucleic acid molecules are present on the pre-implanted solid support. During the templated reaction, one or more supports are typically still in fluid communication.

[0186] Since the pre-implantation reaction can be an RPA reaction and / or the templated process can include more than one RPA reaction according to the teachings of the present invention, the method includes sequential RPA reactions. For example, the first RPA reaction can be a pre-implantation reaction, followed by a second RPA templated reaction. In another example, the pre-implantation reaction can be one or more PCR cycles or non-isothermal amplification cycles, followed by one or more templated reactions involving RPA reactions. The RPA reaction can be carried out under the same conditions. However, in an illustrative embodiment, a pre-implantation RPA reaction or an initial template RPA reaction is performed, followed by one or more subsequent template RPA reactions, so that fewer amplification cycles occur compared to the template RPA reaction or the templated reaction that occurs after the initial template RPA reaction. For example, the pre-implantation RPA reaction can be carried out for a shorter time than the templated RPA reaction, and the templated RPA reaction amplifies the template nucleic acid molecules connected to the pre-implanted solid support produced by the pre-implanted RPA reaction. As a non-limiting illustrative example, a pre-implantation RPA reaction or an initial template RPA reaction is performed, followed by one or more template RPA reactions for 2 to 5 minutes to produce one or more, for example, pre-implanted vectors or templated vector populations, which are then subjected to a template RPA reaction that is performed for 10 to 60 minutes. In some of these non-limiting illustrative examples, prior to the templated RPA reaction, reaction components containing template nucleic acids are washed away from the pre-implanted solid support. All other reaction components of the pre-implantation and templated reactions are included in both the pre-implantation and templated reactions, except that the template nucleic acid molecules are in solution.

[0187] In some embodiments, after the templated reaction, the percentage of sites on one or more vectors in the population of vectors that have a substantially monoclonal template nucleic acid molecule attached thereto is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total amplified vectors (i.e., the total vectors contain an empty, polyclonal, or substantially monoclonal population of template nucleic acid molecules). In illustrative embodiments, after the templated reaction, the percentage of sites on one or more vectors in the population of vectors that have a substantially monoclonal template nucleic acid molecule attached thereto is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the total amplified vectors (i.e., the total vectors comprising an empty, polyclonal or substantially monoclonal population of template nucleic acid molecules) recovered from the reaction mixture. In some embodiments, after the templated reaction, the percentage of sites on one or more vectors in the population of vectors that have a substantially monoclonal template nucleic acid molecule attached thereto is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total vectors recovered from the reaction mixture (i.e., the total vectors include vectors that are not attached to the template nucleic acid molecule, and vectors that have a polyclonal or substantially monoclonal population of template nucleic acid molecules). In illustrative embodiments, after the templated reaction, the percentage of sites on one or more vectors in the population of vectors that have a substantially monoclonal template nucleic acid molecule attached thereto is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the total vectors recovered from the reaction mixture (i.e., the total vectors include vectors that are not attached to the template nucleic acid molecules, and vectors that have a polyclonal or substantially monoclonal population of template nucleic acid molecules).

[0188] In some embodiments, at least a portion of the primers hybridizes with a portion of at least one chain of the polynucleotide in the templated reaction mixture. For example, at least a portion of the primers can hybridize with a nucleic acid adapter that is joined to one or both ends of the polynucleotide. In some embodiments, at least a portion of the primers is partially or completely complementary to a portion of the polynucleotide or to a nucleic acid adapter. In some embodiments, primers are compatible with any type of sequencing platform, including chemical degradation platforms, chain termination platforms, synthetic sequencing platforms, pyrophosphate platforms, massively parallel platforms, ion-sensitive platforms, and single-molecule platforms.

[0189] In some embodiments, the primer (e.g., the first, second, or third primer) has a 5' or 3' overhang tail that does not hybridize to a portion of at least one strand of the polynucleotide in the reaction mixture (a tailed primer). In some embodiments, the tailed primer has any length, including between about 1 and 100 nucleotides in length, such as between about 1 and 90, between about 1 and 80 nucleotides in length, between about 1 and 70 nucleotides in length, between about 1 and 60 nucleotides in length, between about 1 and 50 nucleotides in length, between about 1 and 40 nucleotides in length, or between about 1 and 30 nucleotides in length.

[0190] The disclosed method enables the generation of an amplicon population, in certain embodiments, at least some of which comprise an amplified nucleic acid population. The amplified population produced by the method disclosed herein is suitable for various purposes. In some embodiments, the disclosed method (and related compositions, systems, and kits) optionally comprises further analysis and / or manipulation of the amplified population (amplicon). For example, in some embodiments, the number of amplicons presenting certain desired characteristics is detected and optionally quantified. In some embodiments, after amplification, the amplified product is sequenced. The amplified product sequenced can be an amplicon in a substantially monoclonal nucleic acid population. In some embodiments, the disclosed method comprises amplifying a single member of an amplicon population at different sites or on different carriers. Different sites optionally form a part of a site array. In some embodiments, the site in the site array comprises a hole (reaction chamber) on the surface of the isFET array. Optionally, the nucleic acid molecule to be sequenced is placed at the site. The site can comprise a reaction chamber or a hole. The site can be a part of a similar or identical site array. The array can include a two-dimensional array of sites on a surface (e.g., a surface of a flow cell, an electronic device, a transistor chip, a reaction chamber, a channel, and the like), or a three-dimensional array of sites within a matrix or other medium (e.g., a solid, semi-solid, liquid, fluid, and the like). In any one embodiment of the present teachings, the sites are operably coupled to a sensor. The method includes detecting nucleotide incorporation using a sensor. Optionally, the sites and sensors are located in an array of sites coupled to a sensor.

[0191] In any embodiment of the present teachings, after the templating reaction, the templated support has at least 50,000, 75,000, 100,000, 125,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 10 6In some embodiments, after the templating reaction, the templated vectors have between about 50,000 and 500,000 substantially monoclonal template nucleic acid molecules linked to each templated vector, such as between about 50,000 and 400,000 substantially monoclonal template nucleic acid molecules, between about 50,000 and 300,000 substantially monoclonal template nucleic acid molecules, between about 50,000 and 200,000 substantially monoclonal template nucleic acid molecules, or between about 50,000 and 100,000 substantially monoclonal template nucleic acid molecules linked to each templated vector. In illustrative embodiments, after the templating reaction, the templated vectors have between about 100,000 and 400,000 substantially monoclonal template nucleic acid molecules attached to each templated vector, between about 100,000 and 300,000 substantially monoclonal template nucleic acid molecules, between about 100,000 and 200,000 substantially monoclonal template nucleic acid molecules, or between about 150,000 and 300,000 substantially monoclonal template nucleic acid molecules attached to each templated vector.

[0192] In any of the disclosed embodiments, the amplified template nucleic acid molecules on the support are sequenced. The sequencing method may include any suitable sequencing method known in the art. In some embodiments, template nucleic acid molecules that have been amplified according to the teachings of the present invention are used in any nucleic acid sequencing workflow, including by oligonucleotide probe ligation and detection (e.g., SOLiD TM , from Life Technologies, WO 2006 / 084131), probe-anchor junction sequencing (e.g. Complete Genomics TM or Polonator TM ), synthetic sequencing (such as Gene Analyzer and HiSeq TM , from Illumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX, from 454 Life Sciences), ion-sensitive sequencing (e.g., Personalized Genome Machine (PGM TM )、Ion Proton TM Sequencers, Ion S5 and Ion S5 XL, all from Ion Torrent™ Systems, Inc.), single-molecule sequencing platforms (such as HeliScope TM , from Helicos TM), nanopore sequencing by reading individual bases as they pass through the nanopore (e.g., MinION, from Oxford Nanopore Technologies), chemical degradation sequencing, capillary electrophoresis, gel electrophoresis, and any other next-generation, massively parallel sequencing platform.

[0193] In some sequencing devices, such as Ion Torrent Systems, sequencing reactions are performed in microwells on a surface (e.g., a semiconductor chip). An exemplary embodiment of using a bead carrier in a sequencing device comprising a reaction chamber (e.g., a well) is depicted in Figure 8 For example, refer to Figure 8 , an exemplary sequencing device 416 comprises a well array 418. A bead carrier 406 comprising a target polynucleotide 402 to be sequenced is connected to a magnetic bead 410 to form a bead assembly 412 designed to introduce the bead carrier with the connected target polynucleotide into the well. In some embodiments, the magnetic bead 410 is connected to the bead carrier 406 by double-stranded polynucleotide connection. In some cases, a linker portion is hybridized with a portion of the target polynucleotide on the bead carrier 406. In this example, the linker portion is connected to a complementary linker portion on the magnetic bead 410. In another example, a template polynucleotide for forming a target nucleic acid connected to the bead 406 comprises a linker portion connected to the magnetic bead 410. In another example, a template polynucleotide complementary to the target polynucleotide connected to the bead carrier 406 is generated from a primer modified with a linker connected to the magnetic bead 410. In some embodiments, the linker portion connected to the polynucleotide and the linker portion connected to the magnetic bead are complementary to each other and connected to each other. In one example, the linker moiety has affinity and comprises: an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or an immunoreactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigenin antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and an inhibitor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and a protein A; or an oligonucleotide or a polynucleotide and its corresponding complement. In a specific example, the linker moiety attached to the polynucleotide comprises biotin, and the linker moiety attached to the magnetic bead comprises streptavidin.

[0194] like Figure 8As illustrated in the embodiment depicted in , a plurality of bead carriers 404 and a plurality of polynucleotides 402 (target polynucleotides or template polynucleotides) are placed in a solution. The plurality of bead carriers 404 are activated or otherwise prepared to bind to the polynucleotides 402. For example, the bead carrier 404 comprises an oligonucleotide (capture primer) complementary to a portion of one of the plurality of polynucleotides 402. In another example, the bead carrier 404 is modified with a target polynucleotide 402 using a technique such as biotin-streptavidin binding. In a specific embodiment, the hydrophilic bead particles and the polynucleotides are subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). The template polynucleotide will hybridize with the capture primer. The capture primer is extended to form a bead 406 comprising a target polynucleotide attached thereto. Other beads may not yet be attached to the target nucleic acid, and other template polynucleotides may float freely in the solution. A variety of methods can be used to implant the bead carrier and be captured by the magnetic beads. For example, turning to Fig. 9 At 502, the template polynucleotide (B*-A) is captured by the capture probe (B) attached to the bead carrier 510. The capture probe (B) complementary to the template polynucleotide is extended, thereby generating BA*. Optionally, the resulting double-stranded polynucleotide is denatured, thereby removing the template nucleic acid (B*-A) and leaving a single strand (BA*) attached to the bead carrier 510. Fig. 9 As shown at 504 of the present invention, a primer (A) modified with a linker portion (such as biotin) is hybridized to a portion (A*) of a nucleic acid (BA*) attached to a bead carrier 510. Optionally, the primer (A) is extended to form a complementary nucleic acid (AB*). As shown at 506, magnetic beads 512 are introduced into the solution. Magnetic beads 512 contain a linker that is complementary to the linker portion attached to the primer (A). In this example, the linker attached to the primer (A) is biotin, and the magnetic beads 512 are coated with streptavidin. Magnetic beads 512 can be used to clean the solution and to help place the bead carrier 510 and the attached nucleic acid (BA*) into the well of the sequencing device. As shown at 506 Fig. 9 As shown in 508 of , in some cases, 506 denatures the double-stranded polynucleotide so that the nucleic acid (B*-A) is dehybridized from the nucleic acid (BA*) connected to the bead carrier 510. Therefore, the bead carrier 510 is placed in the hole of the sequencing device, and the bead carrier has a single-stranded target nucleic acid (BA*). Alternatively, the linker-modified probe (A) may not be extended to form a complementary polynucleotide of a polynucleotide (BA*) of a certain length. The extension reaction can be performed using polymerase chain reaction (PCR), recombinase polymerase amplification (RPA) or other amplification reactions.

[0195] Transfer back Figure 8In one embodiment using magnetic beads to help place the bead carrier and attached nucleic acid into the well of the sequencing device, the bead assembly 412 is coated on a substrate 416 of the sequencing device including a well 418. In one example, a magnetic field can be applied to the substrate 416 to pull the magnetic beads 410 of the bead assembly 412 toward the well 418. The bead carrier 406 enters the well 418. For example, the magnet can be moved parallel to the surface of the substrate 416, thereby placing the bead carrier 406 in the well 418. The bead assembly 412 is denatured to remove the magnetic beads 410, leaving the bead carrier 406 in the well 418. For example, thermal cycling or an ionic solution can be used to denature the hybridized double-stranded DNA of the bead assembly 412 to release the magnetic beads 410 and the template polynucleotide having the linker portion attached to the magnetic beads 410. Optionally, the target polynucleotide 406 can be amplified as in a templated reaction, while in the hole 418, a bead carrier 414 with multiple copies of the target polynucleotide is provided. Specifically, the bead 414 has a monoclonal population of the target polynucleotide. Such amplification reactions are performed, for example, using polymerase chain reaction (PCR) amplification, recombinant polymerase amplification (RPA) or a combination thereof. In a particular embodiment, an enzyme (such as a polymerase) is present, bound to, or in close proximity to a particle or bead. In an example, a polymerase is present in a solution or in a hole to promote the replication of a polynucleotide. A variety of nucleic acid polymerases can be used in the methods described herein. In an exemplary embodiment, a polymerase can include an enzyme, a fragment or a subunit thereof that can catalyze the replication of a polynucleotide. In another embodiment, a polymerase can be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fusion or otherwise engineered polymerase, a chemically modified polymerase, a synthetic molecule or an analog, a derivative or a fragment thereof. Although the polynucleotides of the bead carrier 414 are shown as being on the surface, the polynucleotides can be extended within the bead carrier 414. Hydrogels and hydrophilic particles with low concentrations of polymers relative to water can contain polynucleotide segments on and throughout the interior of the bead carrier 414, or polynucleotides can be present in holes and other openings. Specifically, in some examples, the bead carrier 414 permits diffusion of enzymes, nucleotides, primers, and reaction products used to monitor the reaction. Large amounts of polynucleotides per particle produce better signals.

[0196] In some embodiments, sequencing comprises extending the template nucleic acid molecule or amplification template nucleic acid molecule, or extending a sequencing primer hybridized to the template or amplification template by incorporating nucleotides using a polymerase. In some embodiments, sequencing comprises contacting the template or extension primer with a sequencing primer, a polymerase, and at least one type of nucleotides to sequence the template or amplification template connected to the carrier. In some embodiments, sequencing comprises contacting the template or amplification template or extension primer with a sequencing primer, a polymerase, and only one type of nucleotides that do not contain an external label or chain termination group. In some embodiments, a sequencing reaction is performed using at least one sequencing primer hybridized to any portion of a polynucleotide construct (including a nucleic acid adapter or a target polynucleotide sequence).

[0197] Return to Figure 8 In one example, a sequencing primer is added to hole 418, or the bead carrier 414 is pre-exposed to the primer before being placed in hole 418. Specifically, the bead carrier 414 includes a bound sequencing primer. The sequencing primer and the polynucleotide form a nucleic acid duplex comprising a polynucleotide (e.g., a template nucleic acid) hybridized with the sequencing primer. The nucleic acid duplex is at least partially a double-stranded polynucleotide. Enzymes and nucleotides are provided to hole 418 to promote detectable reactions, such as nucleotide incorporation. In some embodiments, sequencing involves detecting nucleotide addition. Methods for detecting nucleotide addition include fluorescent emission methods or ion detection methods. For example, a group of fluorescently labeled nucleotides are provided to system 416 and migrated to hole 418. Excitation energy is also provided to hole 418. When the nucleotide is captured by the polymerase and added to the end of the extension primer, the nucleotide label fluoresces, thereby indicating which type of nucleotide is added. In an alternative example, a solution containing a single type of nucleotide is fed in sequence. In response to nucleotide addition, the pH in the local environment of hole 418 can change. This pH change can be detected by an ion sensitive field effect transistor (ISFET). Thus, the pH change can be used to generate an indication of the order of nucleotides that are complementary to the polynucleotide of particle 410.

[0198] In a typical embodiment of ion-based nucleic acid sequencing, nucleotide incorporation is detected by detecting the presence and / or concentration of hydrogen ions generated by a polymerase-catalyzed extension reaction. In one embodiment, template nucleic acid molecules, optionally pre-bound to sequencing primers and / or polymerases, can be loaded into a reaction chamber, followed by repeated cycles of nucleotide addition and washing. In some embodiments, such templates are attached to a support (such as a particle, bead, or the like) as a substantially monoclonal population, and the substantially monoclonal population is loaded into a reaction chamber.

[0199] In each addition step of the cycle, when the next base in the template is the complementary base of the nucleotide added in the solution, the polymerase extends the primer by incorporating the added nucleotide. If there is one complementary base on the template nucleic acid molecule, then one is incorporated; if there are two consecutive complementary bases on the template nucleic acid molecule, then there are two incorporations; if three, then there are three incorporations; etc. In the case of each such incorporation, there are released hydrogen ions, and the template releases hydrogen ion populations to jointly change the local pH of the reaction chamber. The generation of hydrogen ions is monotonically related to the number of continuous complementary bases in the template (and the total number of template molecules participating in the extension reaction with primers and polymerases). Therefore, when there are many continuous identical complementary bases (i.e., homopolymer regions) in the template, the number of hydrogen ions generated and the amplitude of the local pH change are proportional to the number of continuous identical complementary bases. If the next base in the template is not complementary to the added nucleotide, then incorporation will not occur and hydrogen ions will not be released. In some embodiments, after each step of adding nucleotides, an additional step is performed in which an unbuffered wash solution at a predetermined pH is used to remove nucleotides from the previous step to prevent misincorporation in subsequent cycles. In some embodiments, after each step of adding nucleotides, an additional step is performed in which the reaction chamber is treated with a nucleotide destroying agent (such as apyrase) to eliminate any residual nucleotides remaining in the chamber that may produce spurious extensions in subsequent cycles.

[0200] In certain embodiments, the sequencing system comprises a well or a plurality of wells disposed on a sensor pad of an ionic sensor such as a field effect transistor (FET). In some embodiments, the FET is an array of FETs. The FET or array may comprise, for example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7or more FETs. In an embodiment, the system comprises one or more polymer particles loaded into a hole disposed on a sensor pad of an ionic sensor (e.g., FET), or one or more polymer particles disposed in multiple holes on a sensor pad of an ionic sensor (e.g., FET). In an embodiment, the FET is a chemFET or ISFET. "ChemFET" or chemical field effect transistors include field effect transistor types that act as chemical sensors. ChemFETs have a structural analog of a MOSFET transistor, where the charge on the gate electrode is applied chemically. "ISFET" or ion-sensitive field effect transistors are used to measure ion concentrations in a solution; when the ion concentration (e.g., H+) changes, the current through the transistor changes accordingly. In some embodiments, one or more microfluidic structures are fabricated on an array of FET sensors to provide accommodation or constraints for biological or chemical reactions. For example, in one embodiment, the microfluidic structure is configured to be disposed on one or more holes (or holes, or reaction chambers, or reaction holes, because the terms are used interchangeably herein) above one or more sensors of the array, so that one or more sensors disposed on a given hole detect and measure the presence, level, or concentration of an analyte in a given hole. In an embodiment, there may be a 1:1 correspondence between a FET sensor and a reaction hole.

[0201] return Figure 8 In another example, the wells 418 of the well array are operably connected to a measuring device. For example, for a fluorescence emission method, the wells 418 can be operably coupled to a light detection device. In the case of ionic detection, the lower surface of the wells 418 can be placed on a sensor pad of an ionic sensor (such as a field effect transistor).

[0202] In some embodiments, different classes of nucleotides are added to the reaction chamber sequentially so that each reaction is exposed to different nucleotides one at a time. For example, nucleotides are added in the following order: dATP, dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, etc.; wherein each exposure is followed by a washing step. Depending on the length of the sequence information required, the cycle can be repeated 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 750 times or more.

[0203] In some embodiments, the methods (and related compositions, systems, and kits) comprise detecting, optionally using a FET, the presence of one or more nucleotide incorporation byproducts at a site of the array. In some embodiments, the methods comprise detecting, optionally using a FET, a pH change occurring within at least one reaction chamber. In some embodiments, the disclosed methods further comprise detecting a change in ion concentration in at least one site due to at least one amplification cycle.

[0204] An exemplary system involving sequencing by detecting the ionic byproducts of nucleotide incorporation is the Ion TorrentPGM TM 、Proton TM or S5 TM Sequencer (Thermo Fisher Scientific), an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions produced as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as a byproduct of nucleotide incorporation that occurs during template-dependent nucleic acid synthesis using a polymerase. Ion Torrent PGM TM 、Proton TM or S5 TM The sequencer detects nucleotide incorporation by detecting the hydrogen ion byproduct of nucleotide incorporation. In some embodiments, the Ion Torrent PGM TM 、Proton TM or S5 TMThe sequencer includes a plurality of template polynucleotides to be sequenced, each template being placed in an individual sequencing reaction well in an array. The wells of the array are each coupled to at least one ion sensor for detecting the release of H+ ions or changes in solution pH produced as a byproduct of nucleotide incorporation. The ion sensor includes a field effect transistor (FET) coupled to an ion-sensitive detection layer for sensing the presence of H+ ions or changes in solution pH. The ion sensor provides an output signal indicating the incorporation of nucleotides, which can be represented as a voltage change, the amplitude of which is related to the H+ ion concentration in an individual well or reaction chamber. Different nucleotide types are continuously flowed into the reaction chamber, and can be incorporated into an extension primer (or polymerization site) by a polymerase in an order determined by the template sequence. For example, a sequencing system containing a fluidic loop is connected to at least two reagent storage tanks, a waste liquid storage tank, and is connected to a biosensor by a fluid path for fluid communication through an inlet. Reagents from the storage tank are driven to the fluidic loop by a variety of methods (including pressure, pumps (e.g., syringe pumps), gravity feed, and similar methods), and are selected by control of a valve. The reagents driven from the fluidic loop pass through a valve that receives a signal from a control system. The control system includes a controller for the valve, which generates a signal for opening and closing by an electrical connection. The control system also includes controllers for other components of the system, such as a washing solution valve and a reference electrode connected thereto by an electrical connection. The incorporation of each nucleotide is accompanied by the release of H+ ions in the reaction wells, and the accompanying changes in the local pH. The release of the H+ ions is registered by the FET of the sensor, and the FET generates a signal indicating that the incorporation of nucleotides has occurred. Nucleotides that are not incorporated during the flow of a specific nucleotide may not generate a signal. The amplitude of the signal from the FET may also be related to the number of nucleotides of a specific type incorporated into the extended nucleic acid molecule, thereby allowing the homopolymer region to be parsed. Therefore, during the operation of the sequencer, multiple nucleotides flow into the reaction chamber and are incorporated into monitoring in multiple holes or reaction chambers, allowing the instrument to simultaneously parse the sequence of multiple nucleic acid templates.

[0205] In some embodiments, the method of downstream analysis comprises sequencing at least some of the amplicon population in parallel. Optionally, multiple templates / amplification templates / extension first primers located at different sites of the array are sequenced in parallel.

[0206] In some embodiments, sequencing comprises binding a sequencing primer to at least two different template nucleic acid molecules or at least two different substantially monoclonal populations of nucleic acid molecules. In some embodiments, sequencing comprises incorporating nucleotides onto the 3′ OH of the sequencing primer using a polymerase. Optionally, incorporation comprises forming at least one nucleotide incorporation byproduct.

[0207] In embodiments of the present disclosure involving the distribution of template nucleic acid molecules into wells of an isFET array and subsequent amplification of the template within the wells of the array, an optional downstream analysis step is performed after amplification to quantify the number of sites or wells containing amplified products. In some embodiments, the products of the nucleic acid amplification reaction are detected to count the number of sites or wells containing amplified templates. In some embodiments, after amplification, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the wells contain a substantially monoclonal population of template nucleic acid molecules.

[0208] In some embodiments, the templated vectors are distributed into individual reaction chambers (e.g., well arrays) for sequencing. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the reaction chambers have templated vectors containing a substantially monoclonal population of template nucleic acid molecules. In some embodiments, between about 25% and 95% of the reaction chambers have templated carriers containing a substantially monoclonal population of template nucleic acid molecules, e.g., between about 25% and 85%, between about 25% and 75%, between about 35% and 95%, between about 35% and 85%, between about 35% and 75%, between about 45% and 95%, between about 45% and 85%, between about 45% and 75%, between about 55% and 95%, between about 55% and 85%, between about 55% and 75%, between about 65% and 95%, between about 65% and 85%, between about 65% and 75%, between about 75% and 95%, between about 75% and 85%, or between about 85% and 95% of the reaction chambers have templated carriers containing a substantially monoclonal population of template nucleic acid molecules. In some embodiments, the individual reaction chambers are wells on a sequencing chip. In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25% of the wells produce low quality results, wherein the low quality wells are determined by the sequencing method.

[0209] In various embodiments, the methods, systems and computer-readable media described herein can be advantageously used to process and / or analyze the data and signals obtained from electronic or charge-based nucleic acid sequencing. In electronic or charge-based sequencing (such as pH-based sequencing), ions (such as hydrogen ions) produced by natural byproducts of nucleotide extension reactions catalyzed by polymerase can be detected to determine nucleotide incorporation events. This can be used to sequence template nucleic acid molecules, which can be, for example, fragments of nucleic acid sequences of interest, and can be directly or indirectly connected to carriers (such as particles, microparticles, beads, etc.) as substantially monoclonal populations of nucleic acid molecules. Samples or template nucleic acids can be operably connected to primers and polymerases, and can be subjected to repeated cycles or "flow" (which can be referred to as "nucleotide flow" in this article, by which nucleotides can be produced and incorporated) and washing of nucleotide addition. Primers can be combined with samples or templates so that whenever a nucleotide complementary to the next base in the template is added, the 3' end of the primer is extended by a polymerase. Then, based on the known sequence of the nucleotide flow and based on the measured output signal of the chemical sensor indicating the ion concentration during each nucleotide flow, the type identity, sequence and quantity of nucleotides associated with the sample nucleic acid present in the reaction zone coupled to the chemical sensor can be determined.

[0210] In some embodiments of the methods for generating one or more substantially monoclonal template nucleic acid populations provided herein, a sufficient number of substantially monoclonal or monoclonal template nucleic acid populations are generated to generate at least 60 million sequence reads, or at least 70 million sequence reads, or at least 80 million sequence reads, or at least 90 million sequence reads, or about 100 million sequence reads, or at least 120 million sequence reads, or at least 140 million sequence reads, or at least 150 million sequence reads of at least 200-400 nucleotides in length on one or more Ion Torrent sequencers. In some embodiments of the methods for generating one or more substantially monoclonal template nucleic acid populations provided herein, a sufficient number of substantially monoclonal or monoclonal template nucleic acid populations are generated to generate between about 60 million and 100 million sequence reads of between about 200 to 400 or about 300 to 400 nucleotides in length on one or more Ion Torrent sequencers. In some embodiments of the methods for generating one or more substantially monoclonal template nucleic acid populations provided herein, a sufficient number of substantially monoclonal or monoclonal template nucleic acid populations are generated to generate about 100 million to 150 million sequence reads of between about 100 to 300 or about 100 to 200 nucleotides in length on one or more Ion Torrent sequencers. In some embodiments of the methods for generating one or more substantially monoclonal template nucleic acid populations provided herein, a sufficient number of substantially monoclonal or monoclonal template nucleic acid populations are generated to generate at least 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GB, 2 GB, 5 GB, 10 GB, or 15 GB of sequencing reads on one or more Ion Torrent sequencers. In a templated reaction, a sufficient number of substantially monoclonal or monoclonal populations can be generated to generate at least 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GB, 2 GB, 5 GB, 10 GB, or 15 GB of sequencing reads on one or more Ion Torrent sequencers. TM AQ20 sequencing reads of at least 100MB, 200MB, 300MB, 400MB, 500MB, 750MB, 1GB, or 2GB can be generated on an Ion Torrent Proton, S5, or S5XL sequencer. As used herein, the term "AQ20" and variations thereof refer to a method for measuring the AQ20 sequence of an Ion Torrent Proton, S5, or S5XL sequencer. TMA specific method for sequencing accuracy in a sequencer. Accuracy can be measured with respect to a Phred-like Q score, which measures accuracy on a logarithmic scale: Q10=90%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50=99.999%. For example, in a specific sequencing reaction, an accuracy metric can be calculated by a prediction algorithm or by actual alignment with a known reference genome. The predicted quality score ("Q" score) can be derived from an algorithm that looks at the inherent properties of the input signal and obtains an extremely accurate estimate of whether a given single base included in the sequencing "read" will be aligned. In some embodiments, such predicted quality scores are suitable for filtering and removing lower quality reads before downstream alignment. In some embodiments, the accuracy of the Phred-like Q score report measuring accuracy on a logarithmic scale is such that: Q10=90%, Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50=99.999%. In some embodiments, the data obtained from a given polymerase reaction is filtered to measure only polymerase reads that measure "N" nucleotides or longer and have a Q score above a certain threshold, such as Q10, Q17, Q100 (referred to herein as an "NQ17" score). For example, a 100Q20 score can indicate the number of reads obtained from a given reaction that are at least 100 nucleotides in length and have a Q score of Q20 (99%) or greater. Similarly, a 200Q20 score can indicate the number of reads that are at least 200 nucleotides in length and have a Q score of Q20 (99%) or greater.

[0211] In some embodiments, accuracy is calculated based on appropriate alignment using a reference genome sequence, referred to herein as "raw" accuracy. This is a one-way accuracy that involves measuring the "true" per-base error associated with a single read, as opposed to consensus accuracy, which measures the error rate with a consensus sequence as a result of multiple reads. The raw accuracy measurement can be found in the "AQ" score (for Compare right quality In some embodiments, the data obtained from a given polymerase reaction is filtered to measure only polymerase reads that measure "N" nucleotides or longer, with an AQ score above a certain threshold, such as AQ10, AQ17, AQ100 (referred to herein as "NAQ17" score). For example, a 100AQ20 score may indicate the number of reads obtained from a given polymerase reaction that are at least 100 nucleotides in length and have an AQ score of AQ20 (99%) or greater. Similarly, a 200AQ20 score may indicate the number of reads that are at least 200 nucleotides in length and have an AQ score of AQ20 (99%) or greater.

[0212] In some embodiments, the present disclosure provides a kit for performing nucleic acid amplification in a pre-implantation reaction followed by a templated reaction. The compositions discussed herein are also subject to a kit format, wherein the primers and amplification components can be in the same container, in a separate container, and in a liquid or dehydrated form. The kit may include instructions for performing a method for amplifying template nucleic acid molecules, the method including a pre-implantation reaction for a downstream sequencing method. In one embodiment, the kit provides instructions for preparing for nucleic acid sequencing.

[0213] In certain embodiments, a test kit comprises at least two containers, at least one of which comprises a primer and at least one of which comprises a recombinase. The recombinase and the primer can be in the same or different tubes. In certain embodiments, at least one primer is connected to one or more carriers. The test kit can further comprise one or more pre-implanted solid carriers.

[0214] In some embodiments, the container comprising the recombinase further comprises one or more amplification reagents, which comprise a recombinase auxiliary protein, a polymerase, dNTPs and a buffer. In certain embodiments, the kit comprises one or more containers having: uvsX recombinase, uvsY recombinase load protein, gp32 protein, Sau DNA polymerase, dNTPs, ATP, creatine phosphate and creatine kinase.

[0215] In some embodiments, the kit comprises any combination of: one or more solid supports, polynucleotides, recombinases, recombinase load proteins, single-stranded binding proteins (SSBs), polymerases, nucleotides, ATP, creatine phosphate, creatine kinase, hybridization solutions, washing solutions, buffers, and / or cations (e.g., divalent cations), optionally with a population of at least one primer connected. The kit may comprise all or some of these components, typically in at least two separate containers. The kit may comprise any of the components of the pre-implanted reaction mixture and / or templated reaction mixture.

[0216] Method for generating a nucleic acid template having a specific sequence or a population of two or more nucleic acid templates comprising a specific sequence

[0217] Certain embodiments of the methods, compositions, kits, systems, and devices provided herein include methods for generating a nucleic acid template having a specific nucleotide sequence. In one embodiment, a method for generating a nucleic acid template having a specific nucleotide sequence includes: (a) obtaining a population of nucleic acid molecules, in which each nucleic acid comprises a first continuous nucleotide sequence at the 5' end of the molecule, a second continuous nucleotide sequence at the 3' end of the molecule, and a third nucleotide sequence positioned between the first nucleotide sequence and the second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are different from each other, and wherein in the population, the first nucleotide sequence of the nucleic acid molecules is substantially the same and the second nucleotide sequence of the nucleic acid molecules is substantially the same; (b) subjecting the nucleic acid molecule population to a nucleic acid reaction in the presence of a forward primer and a reverse primer; amplification cycles, wherein the forward primer comprises an oligonucleotide sequence substantially identical to the first nucleotide sequence, the reverse primer comprises an oligonucleotide sequence complementary to a subsequence at the 5' end of the second nucleotide sequence, and the oligonucleotide sequence is connected at the 3' end of the subsequence to a fourth nucleotide sequence that is not complementary to the second nucleotide sequence; in the presence of forward and reverse primers, subjecting the products of the amplification cycles of (b) to amplification cycles to produce a plurality of different nucleic acid products; wherein the reverse primer comprises a nucleotide sequence complementary to the 5' end of the second nucleotide sequence but does not contain a nucleotide sequence complementary to the 3' end of the second nucleotide sequence, and only one of the products comprises a nucleotide sequence complementary to the fourth nucleotide sequence.

[0218] In another embodiment, a method for generating a nucleic acid template having a specific nucleotide sequence comprises: (a) obtaining a nucleic acid containing a nucleic acid chain having a first continuous nucleotide sequence at the 5' end of the nucleic acid chain, a second continuous nucleotide sequence at the 3' end of the nucleic acid chain, and a third nucleotide sequence positioned between the first continuous nucleotide sequence and the second continuous nucleotide sequence, wherein the first continuous nucleotide sequence and the second continuous nucleotide sequence are different from each other; (b) subjecting the nucleic acid to a nucleic acid amplification cycle in the presence of a first primer and a second primer, wherein the first primer comprises a nucleotide sequence substantially identical to the first continuous nucleotide sequence, and the second primer comprises :(i) a nucleotide sequence complementary to the portion of the second continuous nucleotide sequence at the 5' end of the second continuous nucleotide sequence; and (ii) a fourth nucleotide sequence that is not complementary to the second continuous nucleotide sequence and is connected to a sequence complementary to the portion of the second continuous nucleotide sequence at the 3' end of the complementary sequence, and the second primer does not contain a nucleotide sequence complementary to the 3' end of the second continuous nucleotide sequence; and (c) in the presence of the first primer and the second primer, subjecting the product of the nucleic acid amplification cycle of (b) to nucleic acid amplification cycles to produce a plurality of different nucleic acid products, wherein only one of the products contains a nucleotide sequence complementary to the fourth nucleotide sequence. In some embodiments, the method further includes subjecting the product of the nucleic acid amplification cycle of (c) to one or more nucleic acid amplification cycles to produce additional nucleic acid products containing a nucleotide sequence complementary to the fourth nucleotide sequence in the presence of the first primer and the second primer.

[0219] In some embodiments, a method for generating a population of two or more nucleic acid templates having a specific sequence comprises: (a) obtaining an initial nucleic acid population, in which each nucleic acid contains a nucleic acid chain having a first continuous nucleotide sequence at the 5' end of the nucleic acid chain, a second continuous nucleotide sequence at the 3' end of the nucleic acid chain, and a third nucleotide sequence positioned between the first continuous nucleotide sequence and the second continuous nucleotide sequence, wherein the first continuous nucleotide sequence and the second continuous nucleotide sequence are different from each other, and wherein in the population, the first continuous nucleotide sequence is substantially the same, and in the population, the second continuous nucleotide sequence is substantially the same; (b) subjecting the initial nucleic acid population to a nucleic acid amplification cycle in the presence of a first primer and a second primer, wherein the first primer comprises a nucleotide sequence substantially the same as the first continuous nucleotide sequence, and the The second primer comprises: (i) a nucleotide sequence complementary to the portion of the second continuous nucleotide sequence at the 5' end of the second continuous nucleotide sequence; and (ii) a fourth nucleotide sequence that is not complementary to the second continuous nucleotide sequence and is connected to the sequence complementary to the portion of the second continuous nucleotide sequence at the 3' end of the complementary sequence, and the second primer does not contain a nucleotide sequence complementary to the 3' end of the sequence of the two continuous nucleotides; (c) in the presence of the first primer and the second primer, the product of the nucleic acid amplification cycle of (b) is subjected to nucleic acid amplification cycles to produce a plurality of different nucleic acid products, wherein only one of the multiple different nucleic acid products of nucleic acid amplification of each individual nucleic acid in the initial nucleic acid population in step (a) comprises a nucleotide sequence complementary to the fourth nucleotide sequence, thereby producing a population of different nucleic acids, wherein each nucleic acid comprises a nucleotide sequence complementary to the fourth nucleotide sequence. In some embodiments, the method further comprises subjecting the product of the nucleic acid amplification cycle of (c) to one or more nucleic acid amplification cycles in the presence of the first primer and the second primer to produce additional nucleic acid products containing a nucleotide sequence complementary to the fourth nucleotide sequence.

[0220] In some embodiments of the method for producing a nucleic acid template with a specific nucleotide sequence or a population of two or more nucleic acid templates with a specific nucleotide sequence, a forward primer or a first primer comprises a modified nucleotide connected thereto. In certain embodiments, the connection with modified nucleotides comprises a linker portion, such as biotin. In certain embodiments, a forward primer or a first primer comprises a 3' end nucleotide sequence substantially identical to the first nucleotide sequence, a 5' end nucleotide sequence and a non-replicable portion between the 3' end nucleotide sequence and the 5' end nucleotide sequence. In the embodiment of a forward primer or a first primer in which a forward primer or a first primer is included in a non-replicable portion between the 3' end nucleotide sequence substantially identical to the first nucleotide sequence and the 5' end nucleotide sequence, only a product from final nucleic acid amplification comprising a nucleotide sequence complementary to the fourth nucleotide sequence is also included in a single-stranded region of a 5' end nucleotide sequence comprising a non-replicable portion and the first primer at the 5' end. In some embodiments, the method further comprises combining or contacting the single-stranded nucleic acid of the product of the final amplification cycle with a single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence under annealing conditions, thereby hybridizing the product comprising a nucleotide sequence complementary to the fourth nucleotide sequence with the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence to produce a partially double-stranded nucleic acid. In some embodiments, the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence is connected to one or more or more carriers. In some embodiments, the carrier is a solid carrier. In a specific embodiment, the carrier is a particle or a bead. In some embodiments, the method further comprises extending the 3' end of the oligonucleotide portion of the partially double-stranded nucleic acid, thereby generating an extended double-stranded nucleic acid by synthesizing a nucleic acid chain comprising a single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence and having a nucleotide sequence complementary to the product hybridized therewith. In some embodiments, the nucleic acid chain comprising the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence is connected to the carrier by the portion of the oligonucleotide sequence of the chain at the 5' end of the chain. In some embodiments, the method further comprises separating the nucleic acid chain connected to the carrier by collecting the carrier and / or removing it from any other nucleic acid and / or reaction component that is not bound to the carrier.

[0221] In certain aspects, a method for producing a nucleic acid molecule having a specific nucleotide sequence is provided, the method comprising: obtaining a population of nucleic acid molecules, in which each molecule comprises a nucleic acid chain having a first contiguous nucleotide sequence at the 5' end of the molecule, a second contiguous nucleotide sequence at the 3' end of the molecule, and a third nucleotide sequence positioned between the first contiguous nucleotide sequence and the second contiguous nucleotide sequence, wherein the first contiguous nucleotide sequence and the second contiguous nucleotide sequence are different from each other, and wherein in the population, the first contiguous nucleotide sequence is substantially identical, and in the population, the second contiguous nucleotide sequence is substantially identical in the population; and subjecting the population of nucleic acid molecules to two or more cycles of nucleic acid amplification in the presence of one or more forward primers and reverse primers, the one or more forward primers comprising an oligonucleotide sequence substantially identical to the first contiguous nucleotide sequence, the reverse primer being capped at the 3' end and comprising an oligonucleotide sequence complementary to the second contiguous nucleotide sequence, the oligonucleotide sequence being linked at the 5' end of the oligonucleotide sequence to a fourth nucleotide sequence that is not complementary to the second contiguous nucleotide sequence, to produce nucleic acid products, wherein substantially all or all of the products comprise a nucleotide sequence complementary to the fourth nucleotide sequence. In some embodiments, the forward primer comprises a modified nucleotide connected thereto. In some embodiments, the connection to the modified nucleotide comprises a linker portion, such as biotin. In some embodiments, the method further comprises exposing the single-stranded nucleic acid of the amplified product to a single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence under annealing conditions, thereby hybridizing the single-stranded nucleic acid of the product comprising a nucleotide sequence complementary to the fourth nucleotide sequence with the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence to produce a partially double-stranded oligonucleotide binding product. In some embodiments, the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence is connected to a carrier. In some embodiments, the carrier is a solid carrier. In certain embodiments, the carrier is a particle or a bead. In some embodiments, the method further comprises extending the 3' end of the oligonucleotide hybridized with the product comprising a nucleotide sequence complementary to the fourth nucleotide sequence, thereby producing a double-stranded nucleic acid by synthesizing a nucleic acid chain, the nucleic acid chain comprising the single-stranded oligonucleotide substantially identical to the fourth nucleotide sequence and having a nucleotide sequence complementary to the product hybridized therewith. In some embodiments, the method further comprises separating the chain of double-stranded nucleic acids. In some embodiments, the nucleic acid strand comprising the single stranded oligonucleotide substantially identical to the fourth nucleotide sequence is linked to a support at the 5' end of the strand via the oligonucleotide sequence portion of the strand.In some embodiments, said further comprises separating said nucleic acid strands attached to said vector by removing said vector from any other nucleic acids not associated with said vector.

[0222] Method for pre-implanting or implanting a vector with a template nucleic acid

[0223] Certain embodiments of the methods, compositions, kits, systems and devices provided herein include methods of pre-implanting or implanting a vector with a template nucleic acid. In some embodiments, the pre-implantation (or implantation) methods provided herein include any method described herein for generating a nucleic acid template having a specific sequence or a population of two or more nucleic acid templates comprising a specific sequence, wherein the product of one or more of the methods is connected to the vector, for example, by hybridization, for example, by hybridization with a primer on the vector.

[0224] In some embodiments of the method for pre-implanting or implanting a carrier with nucleic acid provided herein, in the presence of one or more or more carriers with capture primers, one or more or more target polynucleotides are amplified. Such methods are included in the presence of a carrier, amplifying a target nucleic acid, the target nucleic acid having a first primer portion, a target portion and a second primer portion, the carrier having a capture primer, a first primer modified by a linker complementary to the first primer portion and a second primer having a portion complementary to at least a portion of the second primer portion, the second primer having a capture primer portion engaged with the portion and complementary to the capture primer, wherein the bead carrier capture primer is extended to include the sequence of the target nucleic acid. In some embodiments, amplification includes performing two or more, or three polymerase chain reaction (PCR) cycles.

[0225] In one embodiment, the method for pre-implanting or implanting a carrier comprises (a) obtaining a nucleic acid containing a nucleic acid chain having a first continuous nucleotide sequence at the 5' end of the nucleic acid chain, a second continuous nucleotide sequence at the 3' end of the nucleic acid chain, and a third nucleotide sequence positioned between the first continuous nucleotide sequence and the second continuous nucleotide sequence; (b) subjecting the nucleic acid to a nucleic acid amplification cycle in the presence of a capture primer, wherein the capture primer comprises a nucleotide sequence complementary to the second continuous nucleotide sequence and is connected to the carrier; (c) subjecting the nucleic acid amplification product of (b) to a nucleic acid amplification cycle in the presence of a capture primer not connected to the carrier and a first primer comprising a nucleotide sequence substantially identical to the first continuous nucleotide sequence. In some embodiments, the first primer is connected to a linker portion.

[0226] Methods and compositions for producing one or more populations of template nucleic acids on a support

[0227] In one aspect, a templated reaction mixture is provided, the templated reaction mixture comprising a pre-implanted solid carrier population, nucleotides, a recombinase, and a polymerase, wherein the pre-implanted solid carrier population comprises between 10 and 50,000 substantially monoclonal template nucleic acid molecules, the template nucleic acid molecules comprising a first primer connected thereto, and the pre-implanted solid carrier population further comprises a first primer connected that is not bound to the template nucleic acid molecules, wherein the reaction mixture does not include a cation capable of starting a recombinase-polymerase amplification reaction, and wherein at least 95% of the template nucleic acid molecules in the reaction mixture are connected to one or more pre-implanted solid carriers. In some embodiments, the template nucleic acid molecules include two or more template nucleic acid molecules with different sequences. In some embodiments, the substantially monoclonal template nucleic acid molecules include a proximal section having 20 to 50 identical nucleotides connecting the template nucleic acid molecules to the solid carrier. In some embodiments, the substantially monoclonal template nucleic acid molecules include less than 100 identical nucleotides at the proximal section connecting the template nucleic acid molecules to the solid carrier. In some embodiments, the pre-implanted solid carrier is a pre-implanted bead. In some embodiments, each pre-implanted solid carrier in the pre-implanted solid carrier population has between 10 and 10,000 substantially monoclonal template nucleic acid molecules connected thereto, and wherein the one or more pre-implanted solid carriers are beads. In some embodiments, the substantially monoclonal template nucleic acid molecules on each pre-implanted solid carrier less than 10% include 50-100 identical nucleotides connected to the pre-implanted solid carrier. In some embodiments, the templated reaction mixture further includes a recombinase auxiliary protein. In some embodiments, the recombinase auxiliary protein is a single-stranded binding protein and / or a recombinase load protein. In some embodiments, a pre-implanted solid carrier is produced using a first recombinase-polymerase amplification (RPA) reaction. In some embodiments, the first RPA reaction is performed by incubating the RPA reaction mixture at a temperature between 35°C and 45°C for 2 to 5 minutes. In some embodiments, the templated reaction mixture further includes the same second primer population in the form of a solution, and wherein the template nucleic acid molecule includes a primer binding site of the first primer at or near the first end. In some embodiments, the reaction mixture further includes a cation capable of starting a recombinase-polymerase amplification reaction.

[0228] One embodiment of the method provided herein for producing one or more templated vectors comprises: (a) forming a templated reaction mixture by combining one or more preimplanted vectors, nucleotides, a recombinase, and a polymerase, wherein the one or more preimplanted vectors comprise a population of substantially identical first primers linked thereto and have a substantially monoclonal template nucleic acid molecule linked thereto, wherein the one or more preimplanted vectors are formed in a separate preimplantation reaction that is performed prior to the templated reaction and comprises the preimplan...

Claims

1. A method for producing a plurality of monoclonal template nucleic acid populations on a carrier, the method comprising: (a) obtaining a plurality of carriers, wherein each carrier has a plurality of single-stranded oligonucleotide primers fixed to the carrier and a template nucleic acid linked to the carrier, wherein the nucleic acid comprises a linked nucleic acid chain having a continuous nucleotide sequence at one end of the chain, the continuous nucleotide sequence being an oligonucleotide primer sequence, in: (i) each vector in the plurality of vectors has a different double-stranded template nucleic acid linked to the vector, (ii) each double-stranded template nucleic acid comprises a linked strand directly linked to the carrier and a strand hybridized to the linked strand, and (iii) each double-stranded template nucleic acid includes a linker portion on the strand hybridized to the joined strand or a single-stranded overhang nucleotide sequence on the strand hybridized to the joined strand; (b) forming a plurality of captured carriers by contacting the plurality of carriers of step (a) with a capture portion bound to the linker portion or with an oligonucleotide complementary to the single-stranded overhang nucleotide sequence and the capture portion, wherein the oligonucleotide complementary to the single-stranded overhang nucleotide sequence hybridizes to the single-stranded overhang and includes a linker portion bound to the capture portion; (c) collecting the captured vector; and (d) separating the support to which the template nucleic acid is attached from the capture moiety or the oligonucleotide complementary to the nucleotide sequence of the single-stranded overhang; (e) subjecting the template nucleic acid ligated to the plurality of carriers to a first isothermal nucleic acid amplification in the presence of (i) a first primer in solution ligated to a linker moiety and (ii) a capture moiety bound to the linker moiety or an oligonucleotide complementary to the single-stranded overhang nucleotide sequence and the capture moiety, wherein the first primer comprises a nucleotide sequence complementary to a primer binding sequence of the ligated nucleic acid strand at the end of the strand opposite to the end having a continuous nucleotide sequence, the continuous nucleotide sequence being the oligonucleotide primer sequence; and (f) subjecting the template nucleic acids linked to the plurality of carriers to a second isothermal nucleic acid amplification in the presence of the first primer in solution with or without a linker moiety and in the absence of a capture moiety linked to the linker moiety or an oligonucleotide complementary to the single-stranded overhang nucleotide sequence and the capture moiety, thereby generating a population of monoclonal template nucleic acids linked to the carriers; The plurality of vectors in step (a) are obtained by a method comprising: (A) obtaining an initial nucleic acid population, in which each nucleic acid includes a nucleic acid chain having a first continuous nucleotide sequence at the 5' end of the chain, a second continuous nucleotide sequence at the 3' end of the chain, and a third nucleotide sequence positioned between the first continuous nucleotide sequence and the second continuous nucleotide sequence, wherein the first continuous nucleotide sequence and the second continuous nucleotide sequence are different from each other, and wherein in the population, the first continuous nucleotide sequence of the nucleic acid population is the same and the second continuous nucleotide sequence of the nucleic acid population is the same; (B) subjecting the nucleic acid population to cycles of nucleic acid amplification in the presence of a first primer and a second primer, in: The first primer comprises a nucleotide sequence identical to the first contiguous nucleotide sequence and is linked to a linker portion, The second primer includes: (i) a nucleotide sequence complementary to a portion of the second continuous nucleotide sequence at the 5' end of the second continuous nucleotide sequence; and (ii) a fourth nucleotide sequence that is not complementary to the second continuous nucleotide sequence and is linked to a sequence complementary to the portion of the second continuous nucleotide sequence at the 3' end of the complementary sequence, and The second primer does not contain a nucleotide sequence complementary to the 3' end of the second continuous nucleotide sequence; (C) subjecting the products of the amplification cycles of step (B) to nucleic acid amplification cycles in the presence of the first primer and the second primer to produce a plurality of different nucleic acid products, wherein only one nucleic acid product of the plurality of different nucleic acid products of nucleic acid amplification of each individual nucleic acid in the initial nucleic acid population in step (A) comprises a nucleotide sequence complementary to the fourth nucleotide sequence and a linker portion; (D) contacting the single strand of the nucleic acid product of step (C) with a carrier to which a plurality of single-stranded oligonucleotide primers identical to the fourth nucleotide sequence are immobilized under annealing conditions, thereby hybridizing the single strand of the nucleic acid product of step (C) comprising a nucleotide sequence complementary to the fourth nucleotide sequence and a linker portion with the immobilized single-stranded oligonucleotide primer to produce a carrier connected to the template nucleic acid comprising a partially double-stranded nucleic acid; and (E) extending the 3′ end of the immobilized oligonucleotide primer portion of the partially double-stranded nucleic acid by template-dependent nucleic acid synthesis to generate an extended double-stranded nucleic acid including a linker portion, thereby generating a plurality of vectors to which the template nucleic acid is linked, wherein each vector has a different double-stranded template nucleic acid linked thereto. 2 . The method of claim 1 , wherein the first isothermal nucleic acid amplification and the second isothermal nucleic acid amplification are performed in a single continuous liquid phase in a single reaction mixture. 3 . The method of claim 1 , wherein each of the plurality of vectors of step (a) has only one double-stranded template nucleic acid ligated thereto.

4. The method of claim 1, wherein the number of vectors contacted with the single strand of the nucleic acid product of step (C) in step (D) exceeds the number of nucleic acid products by at least 2 times, or at least 2.5 times, or at least 3 times, or at least 3.5 times, or at least 4 times, or at least 4.5 times, or at least 5 times, or at least 5.5 times, or at least 6 times, or at least 6.5 times, or at least 7 times, or at least 7.5 times, or at least 10 times, or at least 15 times, or at least 20 times, or at least 25 times.

5. The method according to claim 1, wherein: After step (C) and before step (D), subjecting the nucleic acid product to one or more additional cycles of nucleic acid amplification in the presence of the first primer and the second primer, and / or After step (E), the plurality of carriers to which the template nucleic acid is attached are subjected to one or more cycles of nucleic acid amplification in the presence of the first primer and the second primer and carriers to which a plurality of single-stranded oligonucleotide primers identical to the fourth nucleotide sequence are immobilized. 6 . The method of claim 1 , wherein the plurality of carriers to which the template nucleic acid is attached are contacted with a capture moiety bound to the linker moiety, thereby forming a plurality of captured carriers.

7. A method according to claim 6, wherein the capture portion is connected to a bead or particle, and after collecting the captured carriers, the carrier connected to the template nucleic acid is separated from the capture portion connected to the bead or particle by dissociating the chain of the double-stranded template nucleic acid, thereby producing a plurality of carriers connected to the single-stranded template nucleic acid. 8 . The method according to claim 7 , wherein the carrier linked to the single-stranded nucleic acid is transferred to a surface including microwells and loaded into individual microwells before the first isothermal nucleic acid amplification.

9. A method for producing a plurality of monoclonal template nucleic acid populations on a carrier, the method comprising: (a) obtaining a plurality of carriers, wherein each carrier has a plurality of single-stranded oligonucleotide primers fixed to the carrier and a template nucleic acid linked to the carrier, wherein the nucleic acid comprises a linked nucleic acid chain having a continuous nucleotide sequence at one end of the chain, the continuous nucleotide sequence being an oligonucleotide primer sequence, in: (i) each vector in the plurality of vectors has a different double-stranded template nucleic acid linked to the vector, (ii) each double-stranded template nucleic acid comprises a linked strand directly linked to the carrier and a strand hybridized to the linked strand, and (iii) each double-stranded template nucleic acid includes a linker portion on the strand hybridized to the joined strand or a single-stranded overhang nucleotide sequence on the strand hybridized to the joined strand; (b) forming a plurality of captured carriers by contacting the plurality of carriers of step (a) with a capture portion bound to the linker portion or with an oligonucleotide complementary to the single-stranded overhang nucleotide sequence and the capture portion, wherein the oligonucleotide complementary to the single-stranded overhang nucleotide sequence hybridizes to the single-stranded overhang and includes a linker portion bound to the capture portion; (c) collecting the captured vector; and (d) separating the support to which the template nucleic acid is attached from the capture moiety or the oligonucleotide complementary to the nucleotide sequence of the single-stranded overhang; (e) subjecting the template nucleic acid attached to the plurality of carriers to a first isothermal nucleic acid amplification in the presence of (i) a first primer attached to a linker portion in solution and (ii) a capture portion attached to the linker portion or an oligonucleotide complementary to the single-stranded overhanging nucleotide sequence and the capture portion, wherein the first primer comprises a nucleotide sequence complementary to a primer binding sequence of the attached nucleic acid strand at the end of the strand opposite to the end having the contiguous nucleotide sequence, the contiguous nucleotide sequence being the oligonucleotide primer sequence; and (f) subjecting the template nucleic acids linked to the plurality of carriers to a second isothermal nucleic acid amplification in the presence of the first primer in solution with or without a linker moiety and in the absence of a capture moiety linked to the linker moiety or an oligonucleotide complementary to the single-stranded overhanging nucleotide sequence and the capture moiety, thereby generating a population of monoclonal template nucleic acids linked to the carriers; The plurality of vectors in step (a) are obtained by a method comprising: (A) obtaining an initial nucleic acid molecule population, in which each nucleic acid comprises a nucleic acid chain having a first continuous nucleotide sequence at the 5' end of the chain, a second continuous nucleotide sequence at the 3' end of the chain, and a third nucleotide sequence positioned between the first nucleotide sequence and the second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are different from each other, and wherein in the population, the first nucleotide sequence of the nucleic acid molecule population is the same and the second nucleotide sequence of the nucleic acid molecule population is the same; (B) subjecting the population of nucleic acid molecules to a nucleic acid amplification cycle in the presence of a first primer and a second primer, in: The first primer comprises a 3'-terminal nucleotide sequence and a 5'-terminal nucleotide sequence identical to the first nucleotide sequence, and a non-replicable portion located between the 3'-terminal nucleotide sequence and the 5'-terminal nucleotide sequence. The second primer includes: (i) a nucleotide sequence complementary to a portion of the second nucleotide sequence at the 5' end of the second nucleotide sequence; and (ii) a fourth nucleotide sequence that is not complementary to the second nucleotide sequence and is linked to a sequence complementary to the portion of the second nucleotide sequence at the 3' end of the complementary sequence, and The second primer does not contain a nucleotide sequence complementary to the 3' end of the second nucleotide sequence; (C) subjecting the products of the amplification cycles of step (B) to nucleic acid amplification cycles in the presence of the first primer and the second primer to produce a plurality of different nucleic acid products, wherein only one nucleic acid product among the plurality of different nucleic acid products from the nucleic acid amplification of each individual nucleic acid molecule in the initial nucleic acid molecule population in step (A) comprises a strand having a nucleotide sequence complementary to the fourth nucleotide sequence at the 3' end and a single-stranded region at the 5' end, the single-stranded region comprising the non-replicable portion and the 5' end nucleotide sequence of the first primer; (D) contacting the single strand of the nucleic acid product of step (C) with a carrier to which a plurality of single-stranded oligonucleotide primers identical to the fourth nucleotide sequence are immobilized under annealing conditions, thereby hybridizing the single strand of the nucleic acid product of step (C) including a nucleotide sequence complementary to the fourth nucleotide sequence with the single-stranded oligonucleotide identical to the fourth nucleotide sequence to produce a carrier connected to the template nucleic acid including a partially double-stranded nucleic acid; and (E) extending the 3' end of the fixed oligonucleotide primer portion of the partially double-stranded nucleic acid by template-dependent nucleic acid synthesis to produce an extended double-stranded nucleic acid, wherein the extension of the fixed oligonucleotide primer portion continues to the point where the extended chain hybridizes with the non-replicable portion present on the template chain and is then interrupted, resulting in a partially double-stranded nucleic acid containing a single-stranded overhanging nucleotide sequence comprising the 5' end nucleotide sequence of the first primer, thereby producing a plurality of vectors to which the template nucleic acids are linked, wherein each vector has a different partially double-stranded template nucleic acid linked thereto.

10. The method according to claim 9, wherein: After step (C) and before step (D), subjecting the nucleic acid product to one or more additional cycles of nucleic acid amplification in the presence of the first primer and the second primer, and / or After step (E), the plurality of carriers to which the template nucleic acid is attached are subjected to one or more cycles of nucleic acid amplification in the presence of the first primer and the second primer and carriers to which a plurality of single-stranded oligonucleotide primers identical to the fourth nucleotide sequence are immobilized. 11 . The method according to claim 9 , wherein the nucleotide sequence at the 5′ end of the first primer and the nucleotide sequence at the 3′ end of the first primer are at least complementary to each other.

12. The method according to claim 9, wherein the multiple carriers connected with partially double-stranded template nucleic acids are contacted with oligonucleotides complementary to the single-stranded protruding end nucleotide sequence and a capture portion, wherein the oligonucleotides complementary to the single-stranded protruding end nucleotide sequence form a hybrid by hybridizing with the single-stranded protruding end nucleotide sequence, and include a linker portion to which the capture portion can bind, thereby forming a plurality of captured carriers.

13. The method according to claim 12, wherein the 5' terminal nucleotide sequence of the first primer and the 3' terminal nucleotide sequence of the first primer are at least complementary to each other, and the contacting is performed under the condition that the 5' terminal nucleotide sequence of the first primer and the 3' terminal nucleotide sequence of the first primer do not hybridize to each other.

14. A method according to claim 13, wherein after contacting with an oligonucleotide complementary to the single-stranded protruding end nucleotide sequence, the multiple carriers connected with a partial double-stranded template nucleic acid are contacted with a capture portion, and the contact with the capture portion is carried out under conditions where the 5' end nucleotide sequence of the first primer and the 3' end nucleotide sequence of the first primer can hybridize with each other.

15. A method according to claim 12, wherein the capture portion is connected to a bead or particle, and after collecting the captured carriers, the multiple carriers connected to the template nucleic acid are separated from the capture portion connected to the bead or particle by dissociating the chains of the hybrid of the single-stranded protruding end nucleotide sequence and the oligonucleotide complementary to the single-stranded protruding end nucleotide, thereby producing a plurality of carriers connected to a partially double-stranded template nucleic acid containing a single-stranded protruding end nucleotide sequence. 16 . The method according to claim 15 , wherein the carrier connected to the partially double-stranded template nucleic acid containing the single-stranded overhang nucleotide sequence is transferred to a surface including microwells and loaded into individual microwells before the first isothermal nucleic acid amplification.

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