Amplification compositions and methods

Abstract

The present disclosure relates to novel amplification compositions and methods, in particular for sequencing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 411,949, filed on September 30, 2022, and entitled “Amplification Compositions and Methods,” the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to novel amplification compositions and methods, particularly for use in sequencing.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. The xml copy created on September 22, 2023 is named 85491_08300_PCT.xml and is 16.5 kilobytes in size. Background Art

[0006] Detection of analytes (such as nucleic acid sequences) present in biological samples has been used as a method to identify and classify microorganisms, diagnose infectious diseases, detect and characterize genetic abnormalities, identify genetic changes associated with cancer, study genetic susceptibility to disease, and measure responses to various types of treatments. A common technique for detecting analytes (such as nucleic acid sequences) in biological samples is nucleic acid sequencing.

[0007] Advances in the study of biomolecules have been driven in part by improvements in techniques used to characterize molecules or their biological reactions. In particular, the study of nucleic acids DNA and RNA has benefited from the development of techniques for sequence analysis.

[0008] Nucleic acid amplification methods are known which allow the amplification products to be immobilized on a solid support so as to form an array consisting of clusters or "colonies" formed by a plurality of identical immobilized polynucleotide chains and a plurality of identical immobilized complementary chains. The nucleic acid molecules present in the DNA colonies on the cluster arrays prepared according to these methods can provide templates for sequencing reactions.

[0009] A method for sequencing a polynucleotide template involves performing multiple extension reactions using a DNA polymerase to successively incorporate labeled nucleotides into the template strand. In such a "sequencing by synthesis" reaction, a new nucleotide strand that base pairs with the template strand is constructed in the 5' to 3' direction by successively incorporating individual nucleotides that are complementary to the template strand.

[0010] There remains a need to develop new amplification compositions and methods that increase the throughput and accuracy of sequencing runs. The present disclosure addresses this need. Summary of the invention

[0011] According to one aspect, an amplification composition (also referred to herein as a composition) comprising an inorganic polyphosphate and a polyphosphate kinase is provided.

[0012] Preferably, the amplification composition may comprise at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, and NTP.

[0013] Preferably, the amplification composition comprises a recombinase.

[0014] Preferably, the amplification composition comprises a single-stranded nucleotide binding protein.

[0015] Preferably, the amplification composition comprises a polymerase.

[0016] Preferably, the amplification composition comprises NTPs.

[0017] Preferably, the amplification composition comprises a recombinase, a single-stranded nucleotide binding protein, a polymerase and NTPs.

[0018] Preferably, the amplification composition comprises an inorganic polyphosphate at a concentration of about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 2 μM to about 10 μM.

[0019] Preferably, the amplification composition comprises polyphosphate kinase at a concentration of about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 2 μM to about 10 μM.

[0020] Preferably, the inorganic polyphosphate comprises a first inorganic polyphosphate having less than 50 phosphate residues.

[0021] Preferably, the inorganic polyphosphate comprises a second inorganic polyphosphate having more than 100 phosphate residues.

[0022] Preferably, the ratio of the first inorganic polyphosphate to the second inorganic polyphosphate is from about 90:10 to about 10:90, from about 80:20 to about 20:80, from about 70:30 to about 30:70, from about 60:40 to about 40:60, or about 50:50.

[0023] Preferably, the polyphosphate kinase is a thermophilic polyphosphate kinase.

[0024] Preferably, the polyphosphate kinase has an optimal operating temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

[0025] Preferably, the polyphosphate kinase is selected from the group consisting of a polyphosphate kinase of the PPK1 family, a polyphosphate kinase of the PPK2 family and a polyphosphate kinase of the PPK3 family.

[0026] Preferably, the polyphosphate kinase comprises an amino acid sequence as defined in SEQ ID NOs: 1 to 3, or a functional variant or functional fragment thereof.

[0027] Preferably, the composition does not comprise PEG.

[0028] Preferably, the amplification composition comprises a buffer.

[0029] Preferably, the amplification composition is buffered to a pH of about 6.0 to about 9.0, preferably about 6.5 to about 8.8, more preferably about 7.5 to about 8.7, even more preferably about 8.3 to about 8.6.

[0030] Preferably, the composition is a clustered composition or a sequencing-by-synthesis amplification composition or a resynthesized composition.

[0031] According to another aspect, a kit comprising an inorganic polyphosphate and a polyphosphate kinase is provided.

[0032] Preferably, the kit comprises an amplification composition as described herein.

[0033] Preferably, the kit may comprise at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, and NTP.

[0034] Preferably, the kit comprises a recombinase.

[0035] Preferably, the kit comprises a single-stranded nucleotide binding protein.

[0036] Preferably, the kit comprises a polymerase.

[0037] Preferably, the kit comprises NTPs.

[0038] Preferably, the kit comprises a recombinase, a single-stranded nucleotide binding protein, a polymerase and NTPs.

[0039] Preferably, the kit further comprises a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

[0040] According to another aspect, there is provided use of an amplification composition as described herein or a kit as described herein in amplifying a nucleic acid template or in sequencing a nucleic acid sequence.

[0041] According to another aspect, a method of amplifying a nucleic acid template is provided, wherein the method comprises recycling ADP to ATP using an inorganic polyphosphate and a polyphosphate kinase.

[0042] Preferably, the method comprises adding an amplification composition as described herein.

[0043] Preferably, the method comprises adding a first polyphosphate composition and a second polyphosphate composition, wherein in the first polyphosphate composition, the amount of the first inorganic polyphosphate is relatively high relative to the amount of the second inorganic polyphosphate, and

[0044] Wherein in the second polyphosphate composition, the amount of the second inorganic polyphosphate is relatively high relative to the amount of the first inorganic polyphosphate.

[0045] Preferably, the first polyphosphate composition is added before the second polyphosphate composition.

[0046] Preferably, the amplification is performed by exclusive amplification.

[0047] According to another aspect, a method for sequencing a nucleic acid sequence is provided, wherein the method comprises:

[0048] amplifying the nucleic acid template using the methods described herein; and

[0049] The amplified nucleic acid template is sequenced.

[0050] Preferably, the step of sequencing the amplified nucleic acid template comprises performing a first sequencing read and a second sequencing read.

[0051] Preferably, the step of sequencing the amplified nucleic acid template is performed using a sequencing-by-synthesis technique or a sequencing-by-ligation technique.

[0052] Preferably, the process is carried out isothermally.

[0053] Preferably, the process is conducted at a temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

[0054] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1AA schematic diagram of an exemplary method of sequencing a nucleic acid according to an embodiment of the present disclosure is shown. Figure 1B Shows typical inorganic polyphosphate (PolyP n ) structure, where n is the number of phosphate residues. Figure 1C The use of PolyP catalyzed by PPK1 was shown n Reaction scheme for recycling ADP to form ATP and an inorganic polyphosphate in which the number of phosphate residues has been reduced by 1 (ie, to the number of phosphate residues n-1).

[0056] Figure 2 It is shown how PolyP can be enzymatically converted to ATP in the presence of ADP and the enzyme polyphosphate kinase 1 (PPK1), and how PolyP can be enzymatically converted to ATP in the presence of ADP and the enzyme polyphosphate kinase 1 (PPK2). Inorganic polyphosphates (PolyP) are high-energy phosphoanhydride molecules of varying lengths that can be donors for ATP production. ATP is an essential small molecule in the amplification / clustering process. ATP supports key functions of the recombinase, including filamentation, homology, search, and invasion. ADP is a byproduct of the recombinase enzymatic reaction, and the accumulation of this small molecule can inhibit the amplification / clustering reaction. The removal of ADP promoted by the PPK1 / PPK2 enzyme removes this inhibitory molecule by recycling to produce ATP.

[0057] Figure 3A and Figure 3B The expression and purification of recombinant Thermus thermophilus (Tth) PPK1 and Meiothermus ruber (Mru) PPK2 are shown. Figure 3A - SDS-PAGE analysis of Tth PPK1 and Mru PPK2 expression conditions. Lanes: (M) size markers; (1) uninduced-PPK1; (2) induced 37°C soluble-PPK1; (3) precipitated 37°C-PPK1; (4) induced 18°C ​​soluble-PPK1; (5) precipitated 18°C-PPK1; (6) uninduced-PPK2; (7) induced 37°C soluble-PPK2; (8) precipitated 37°C-PPK2; (9) induced 18°C ​​soluble-PPK2; (10) precipitated 18°C-PPK2. Figure 3B -B-SDS-PAGE analysis of IMAC purification of Mru PPK2. Lanes: (M) size marker; (1) clear soluble material applied to column; (2) FT; (3) wash; (4) elution fraction 1; (5) elution fraction 2; (6) elution fraction 3; (7) elution fraction 4.

[0058] Figure 4It is shown how PPi can be enzymatically converted to ATP in the presence of ADP and the enzyme polyphosphate kinase 1 (PPK1), and how PPi can be enzymatically converted to ATP in the presence of ADP and the enzyme polyphosphate kinase 2 (PPK2).

[0059] Figure 5A .) Sequence Analysis Viewer (SAV) was used to extract read 1 (R1) and read 2 (R2) intensities from the NextSeq 2000 run. The striped black and white bars are the R1 or R2 intensities of the control clustered formulations with standard commercial recipes. The gray bars are the R1 or R2 intensities of the clustered formulations supplemented with 0.3U PPiase (pyrophosphatase) / 100μl using a standard modified recipe to be taken out from a unique well with a box. The black bars are the R1 or R2 intensities of the clustered formulations supplemented with 1.2U PPiase / 100μl using a standard modified recipe to be taken out from a unique well with a box. For read 1 and read 2, the presence of PPiase increases the intensity. In addition, an increase in intensity was observed in a concentration-dependent manner, which means that an increase in the amount of enzyme used increases the intensity signals of read 1 and read 2. The unit of intensity measurement is relative fluorescence unit (RFU). Figure 5B .) Mass fractions expressed as %Q30 values ​​were extracted from SAV. The control bar is a standard clustered formulation; the gray bar is a clustered formulation supplemented with 0.3UPPiase / 100μl clustered formulation; the black bar is a clustered formulation 1.2UPPiase / 100μl clustered formulation. Relative to the control, the %Q30 score increased in the presence of PPiase in a concentration-dependent manner. Figure 5C .) Instrument yield measured as G output was extracted from SAV. Control bars are standard clustering formulations; gray bars are clustering formulations supplemented with 0.3U PPiase / 100μl clustering formulations; black bars are clustering formulations 1.2U PPiase / 100μl clustering formulations. Relative to the control, the yield of the NextSeq 2000 increased in the presence of PPiase in a concentration-dependent manner. Figure 5D .) Extract the percentage of clusters passing the filter (% PF) from SAV. The control bar is the standard clustering formulation; the gray bar is the clustering formulation supplemented with 0.3U PPiase / 100μl clustering formulation; the black bar is the clustering formulation 1.2U PPiase / 100μl clustering formulation. Relative to the control, the % PF of NextSeq 2000 increased in the presence of PPiase in a concentration-dependent manner. Figure 5E .) Addition of inorganic pyrophosphatase as reference means reduction to PPi.

[0060] Figure 6PPK2 and PPi are shown to support in vitro RPA reactions. Lanes: (M) DNA ladder with annotated sizes; (1) positive control of all components of HCXE (normal amplification mixture using creatine kinase (CK) and creatine phosphate (CP)); (2) HCXE + 2.5 μM PPi; (3) HCXE + 2.5 μM PPi + 3 μM PPK2 + 1.8 μM ATP (CK and CP removed); (4) HCXE and 2.5 μM PPi + 1.8 μM ATP (CK and CP removed); (5) negative control, template minus amplification mixture.

[0061] Figure 7 An overview of the strand invasion assay (SIA) is shown. An annealed duplex, one strand (red) of which is attached with a black hole quencher (BHQ) and one strand (black) is attached with a fluorescent dye (FAM). An oligonucleotide (oligo; green) having a region homologous to the annealed duplex (red) is present in a reaction tube. A combination of (+ / -) a recombinase and an energy regeneration system can be supplemented into the reaction tube. When the BHQ strand is removed by the invading unlabeled strand, a fluorescent signal is generated, causing the FAM dye to be excited.

[0062] Fig. 8A .) Engineered RB32 UvsX confers thermostability to a mesophilic enzyme. A.) Alignment showing amino acid differences between engineered RB32. Figure 8B .) SIA fluorescence plots of engineered RB32 over a range of concentrations (from 0.2 μM to 2 μM) at 60°C. Figure 8C .) SIA fluorescence plots of engineered RB32 over a range of concentrations (from 0.2 μM to 2 μM) at 50°C. Fig.8D .) Vmax was plotted for comparison between enzyme concentrations based on fluorescence readings calculated by the instrument software (Biotek; Cytation 5). The concentrations tested are shown in the key parts of the figure. Engineered RB32 UvsX has strand invasion activity at 60°C, compared to the reduced activity of HQ UvsX at 60°C. DETAILED DESCRIPTION

[0063] The features described below apply to all aspects and embodiments of the present disclosure.

[0064] The present disclosure relates to amplification methods and compositions.

[0065] The present disclosure can be used for sequencing, such as paired sequencing. Methods suitable for the present disclosure have been described in WO 08 / 041002, WO 07 / 052006, WO 98 / 44151, WO 00 / 18957, WO 02 / 06456, WO 07 / 107710, WO05 / 068656, US13 / 661,524 and US 2012 / 0316086, the contents of which are incorporated herein by reference. Further information may be found in US20060024681, US200602926U, WO 06110855, WO 06135342, WO 03074734, WO07010252, WO 07091077, WO 00179553 and WO 98 / 44152, the contents of which are incorporated herein by reference.

[0066] Sequencing generally involves four basic steps: 1) library preparation to form multiple template molecules that can be used for sequencing; 2) cluster generation to form an array of amplified single template molecules on a solid support; 3) sequencing the cluster array; and 4) data analysis to determine the target sequence.

[0067] Library preparation is the first step in any high-throughput sequencing platform. During library preparation, nucleic acid sequences (such as genomic DNA samples, or cDNA or RNA samples) are converted into sequencing libraries, and then the nucleic acid sequences can be sequenced. Taking DNA samples as an example, the first step of library preparation is the random fragmentation of DNA samples. First, the sample DNA is fragmented, and the fragments of a specific size (usually 200bp to 500bp, but also larger) are connected, subcloned or "inserted" between two oligonucleotide adapters (adapter sequences). This can be subsequently amplified and sequenced. The initial sample DNA fragment is referred to as "insertion sequence". Alternatively, "labeling" can be used to attach sample DNA to adapters. In labeling, double-stranded DNA is fragmented and labeled with adapter sequences and PCR primer binding sites at the same time. Combinatorial reaction eliminates the need for a separate mechanical shearing step during library preparation. Before being modified with adapter sequences, the target polynucleotides can also be advantageously graded by size.

[0068] As used herein, an "adaptor" sequence comprises a short sequence-specific oligonucleotide that is ligated to the 5' and 3' ends of each DNA (or RNA) fragment in a sequencing library as part of library preparation. An adaptor sequence may also comprise a non-peptide linker.

[0069] As will be understood by the technician, double-stranded nucleic acid will generally be formed by two complementary polynucleotide chains, which are composed of deoxyribonucleotides connected by phosphodiester bonds, but may additionally include one or more ribonucleotides and / or non-nucleotide chemical moieties and / or non-naturally occurring nucleotides and / or non-naturally occurring backbone bonds. Specifically, double-stranded nucleic acid may include non-nucleotide chemical moieties, such as joints or spacers at the 5' ends of one or two chains. As a non-limiting example, double-stranded nucleic acid may include methylated nucleotides, uracil bases, thiophosphate groups, and may also include peptide conjugates, etc. Such non-DNA or non-natural modifications may be included to give nucleic acid some desired characteristics, such as for realizing covalent attachment, non-covalent attachment or metal coordination attachment to a solid support, or serve as a spacer to position the cleavage site to the optimal distance from a solid support. Single-stranded nucleic acid is composed of one such polynucleotide chain. In the case where a polynucleotide chain is only partially hybridized with a complementary chain, for example, a long polynucleotide chain hybridized with a short nucleotide primer, it may still be referred to as a single-stranded nucleic acid herein.

[0070] In one embodiment, the template includes a first primer binding sequence (e.g., P5, e.g., comprising a sequence as defined in SEQ ID NO: 4), an index sequence (e.g., i5), a first sequencing binding site (e.g., SBS3), an insertion sequence, a second sequencing binding site (e.g., SBS12), a second index sequence (e.g., i7), and a second primer binding sequence (e.g., P7', e.g., comprising a sequence as defined in SEQ ID NO: 7) in the 5' to 3' direction. In another embodiment, the template includes a first primer binding site (e.g., P5' complementary to P5, e.g., comprising a sequence as defined in SEQ ID NO: 6), an index sequence (e.g., i5' complementary to I5), a first sequencing binding site (e.g., SBS3' complementary to SBS3), an insertion sequence, a second sequencing binding site (e.g., SBS12' complementary to SBS12), a second index sequence (e.g., i7' complementary to I7), and a second primer binding sequence (e.g., P7' complementary to P7, e.g., comprising a sequence as defined in SEQ ID NO: 5) in the 3' to 5' direction. Either template is referred to herein as a "template strand" or a "single-stranded template". The two template strands that anneal together are referred to herein as a "double-stranded template."

[0071] A sequence comprising at least one primer binding sequence (preferably a combination of a primer binding sequence, an index sequence, and a sequencing binding site) may be referred to herein as an adapter sequence, and a single insert sequence is flanked by a 5' adapter sequence and a 3' adapter sequence. The first primer binding sequence may also include a sequencing primer for index reading (I5). In the present disclosure, a "primer binding sequence" may also be referred to as a "clustered sequence," and such terms may be used interchangeably.

[0072] The P5' primer binding sequence and the P7' primer binding sequence are complementary to the short primer sequence (or lawn primer) present on the surface of the flow cell. The binding of P5' and P7' to their complementary sequences (P5 and P7) on, for example, the surface of the flow cell allows nucleic acid amplification. As used herein, "'" indicates a complementary strand.

[0073] The length of the primer binding sequence in the adapter that allows hybridization with an amplification primer (e.g., moss primer) is generally about 20-40 nucleotides, although in an embodiment, the present disclosure is not limited to sequences of this length. The exact identity in the amplification primer (e.g., moss primer), and therefore the homologous sequence in the adapter is generally unimportant for the present disclosure, as long as the primer binding sequence can interact with the amplification primer to guide PCR amplification. The sequence of the amplification primer can be specific to the specific target nucleic acid desired to be amplified, but in other embodiments, these sequences can be "universal" primer sequences, which can amplify any target nucleic acid with known or unknown sequence, which has been modified to be able to amplify with universal primers. The design criteria for PCR primers are generally well known to those of ordinary skill in the art.

[0074] Index sequences (also called barcodes or tag sequences) are unique short DNA (or RNA) sequences that are added to each DNA (or RNA) fragment during library preparation. Unique sequences allow many libraries to be merged together and sequenced simultaneously. Before final data analysis, sequencing reads from the merged libraries are identified and classified by calculation based on their barcodes. Library multiplexing is also a useful technique when working with small genomes or targeting genomic regions of interest. Multiplexing with barcodes can exponentially increase the number of samples analyzed in a single run without significantly increasing running costs or running time. Examples of tag sequences are found in WO05068656, the contents of which are incorporated herein by reference in their entirety. The tag can be read at the end of the first read, or also at the end of the second read, for example using a sequencing primer complementary to the P7 of the chain marker. The present disclosure is not limited to the number of reads per cluster, for example two reads per cluster: three or more reads per cluster can be obtained simply by dehybridizing the first extended sequencing primer and hybridizing the second primer before or after the cluster re-growth / chain resynthesis step. Methods for preparing suitable samples for indexing are described in, for example, US60 / 899221. Single or dual indexes may also be used. Using a single index, up to 48 unique 6-base indexes may be used to generate a library of up to 48 unique markers. Using a dual index, up to 24 unique 8-base index 1 sequences and up to 16 unique 8-base index 2 sequences may be used in combination to generate a library of up to 384 unique markers. Index pairs may also be used so that each i5 index and each i7 index are used only once. Using these unique dual indexes, it is possible to identify and filter skip reads of the indexes, thereby providing even higher confidence in multiplexed samples.

[0075] The sequencing binding site is a sequencing and / or index primer binding site, and indicates the starting point of sequencing reading. During the sequencing process, a portion of the sequencing binding site on the template strand is annealed (i.e., hybridized) by a sequencing primer. Polymerase binds to the site, and the complementary nucleotides are incorporated into the growing opposite strand one by one. In one embodiment, the sequencing process includes a first sequencing read and a second sequencing read. The first sequencing read may include the first sequencing primer (reading 1 sequencing primer) and the first sequencing binding site (e.g., SBS3'), followed by the synthesis and sequencing of the complementary strand. This results in the sequencing of the inserted sequence. In a second step, an index sequencing primer (e.g., i7 sequencing primer) is bound to the second sequencing binding site (e.g., SBS12), thereby resulting in the synthesis and sequencing of the index sequence (e.g., sequencing of i7 primers). The second sequencing read may include the combination of an index sequencing primer (e.g., i5 sequencing primer) and the complementary sequence (e.g., SBS3) of the first sequencing binding site on the template, and the synthesis and sequencing of the index sequence (e.g., i5). In the second step, a second sequencing primer (read 2 sequencing primer) that binds to the complementary sequence of the primer (eg, i7 sequencing primer) binds to a second sequencing binding site (eg, SBS12'), resulting in the synthesis and sequencing of the insert sequence in the reverse direction.

[0076] Once a double-stranded nucleic acid template library is formed, typically the library has previously been subjected to denaturing conditions to provide single-stranded nucleic acids. Suitable denaturing conditions will be apparent to the skilled artisan, with reference to standard molecular biology protocols (Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY; Current Protocols, Ausubel et al., eds.). In one embodiment, chemical denaturation is used.

[0077] After denaturation, the single-stranded template library can be contacted in free solution to a solid support containing surface capture moieties (e.g., P5 and P7 moss primers.) The solid support is typically a flow cell, although in alternative embodiments, other types of solid supports can be used for seeding and clustering outside of the flow cell.

[0078] As a simple example, after P5 primer and P7 primer are attached to solid support, solid support can be contacted with template to be amplified under the condition of allowing hybridization (or annealing, these terms can be used interchangeably) between template and fixed primer. Usually, template is added in free solution under suitable hybridization conditions, which will be apparent to the technician. Usually, hybridization conditions are, for example, 5xSSC at 40 ℃. However, other temperatures can be used during hybridization, such as about 50 ℃ to about 75 ℃, about 55 ℃ to about 70 ℃ or about 60 ℃ to about 65 ℃. Solid phase amplification can be carried out then. The first step of amplification is primer extension step, wherein nucleotides are added to the 3 ' end of fixed primer using template, to produce a complementary chain extended completely. Then template is usually washed off from solid support. Complementary chain includes a primer binding sequence (i.e. P5 ' or P7 ') at its 3 ' end, which can be bridged to and combined with the second primer molecule fixed on the solid support. Further rounds of amplification (analogous to standard PCR reactions) lead to the formation of (monoclonal) clusters or colonies of template molecules bound to the solid support.

[0079] Therefore, solid phase amplification by a method similar to WO 98 / 44151 or WO 00 / 18957 (the contents of which are incorporated herein by reference in their entirety) will result in a cluster array consisting of colonies of "bridging" amplification products. The two chains of the amplification product will be fixed to the solid support at or near the 5' end, and this attachment is derived from the initial attachment of the amplification primer. Typically, the amplification product in each colony will be derived from the amplification of a single template (target) molecule. Other amplification procedures can be used, and will be known to the technician. For example, amplification can be isothermal amplification using a strand displacement polymerase; or it can be exclusive amplification, as described in WO 2013 / 188582. Further information about amplification can be found in WO0206456 and WO07107710, the contents of which are incorporated herein by reference in their entirety. By such methods, a cluster of a single template molecule is formed.

[0080] To facilitate sequencing, it is preferred that in the event that one of the strands is removed from the surface, this allows efficient hybridization of the sequencing primer to the remaining immobilized strand. Suitable methods for linearization are described in more detail in application number WO07010251, the contents of which are incorporated herein by reference in their entirety.

[0081] By obtaining the sequence read by a strand of the template from the primer in the solution, using the fixed primer to copy the strand, releasing the first strand and sequencing the second replicated strand, sequence data can be obtained from the two ends of the template duplex. For example, sequence data can be obtained from the two ends of the fixed duplex by such a method: wherein the duplex is processed to release the 3'-hydroxyl moiety that can be used as an extension primer. The first sequence can then be read from a strand of the template using an extension primer. After reading for the first time, the strand can be extended to fully replicate all bases until the end of the first strand. The second copy remains attached to the surface of the 5' end. When the first strand is removed from the surface, the sequence of the second strand can be read. This provides sequence readings from the two ends of the original fragment.

[0082] Any suitable "sequencing by synthesis" technique can be used for sequencing, in which nucleotides are continuously added to free 3' hydroxyl groups, resulting in the synthesis of polynucleotide chains in the 5' to 3' direction. The properties of the added nucleotides are preferably determined after each addition. A specific sequencing method relies on the use of modified nucleotides that can act as reversible chain terminators. Such reversible chain terminators include removable 3' blocking groups. Once such modified nucleotides have been incorporated into the growing polynucleotide chain complementary to the template region to be sequenced, there is no free 3'-OH group available to guide further sequence extension, so the polymerase cannot add additional nucleotides. Once the properties of the bases incorporated into the growing chain have been determined, the 3' block can be removed to allow the addition of the next continuous nucleotide. By sorting the products derived from these modified nucleotides, the DNA sequence of the DNA template can be inferred. If each of the modified nucleotides has been attached with a different label known to correspond to a specific base, it is beneficial to distinguish the bases added at each incorporation step, then such reactions can be completed in a single experiment. Suitable labels are described in PCT application PCT / GB / 2007 / 001770, the contents of which are incorporated herein by reference in their entirety. Alternatively, separate reactions containing each modified nucleotide added separately may be performed.

[0083] Modified nucleotides can carry labels to facilitate their detection. In a specific embodiment, the label is a fluorescent label. Each nucleotide type can carry different fluorescent labels. However, detectable labels do not have to be fluorescent labels. Any label that allows detection of nucleotide incorporation into a DNA sequence can be used. A method for detecting fluorescently labeled nucleotides includes using a laser with a specific wavelength for the labeled nucleotide, or using other suitable illumination sources. The fluorescence from the label on the incorporated nucleotide can be detected by a CCD camera or other suitable detection methods. Suitable detection methods are described in PCT / US2007 / 007991, the contents of which are incorporated herein by reference in their entirety.

[0084] Alternative sequencing methods include sequencing by ligation, for example as described in US6306597 or WO06084132, the contents of which are incorporated herein by reference.

[0085] In some embodiments, sequencing may involve paired sequencing. Typical steps for paired sequencing are known and described in WO 2008 / 041002, the contents of which are incorporated herein by reference. However, the key steps will be briefly described.

[0086] The examples disclosed herein relate to methods for sequencing two regions of a target double-stranded polynucleotide template (referred to herein as the first region and the second region for sequence determination). The first region and the second region for sequence determination are located at the two ends of the complementary strand of the double-stranded polynucleotide template, and they are referred to herein as the first template strand and the second template strand, respectively. Once the sequence of a known strand, the sequence of its complementary strand is also known, so the term two regions can be equally applicable to the two ends of a single-stranded template or the two ends of a double-stranded template, wherein the first region and its complement are known, and the second region and its complement are known.

[0087] A plurality of template polynucleotide duplexes are fixed on a solid support. The template polynucleotide can be fixed in the form of a single template molecule array or "cluster" of amplification. Each duplex in the duplex in a specific cluster contains the same double-stranded target region to be sequenced. These duplexes are each formed by a complementary first template strand and a second template strand, which are connected to a solid support at or near their 5' ends. Typically, the template polynucleotide duplex will be provided in the form of a cluster array.

[0088] Another starting point is multiple single-stranded templates attached to the same surface as multiple primers complementary to the 3' end of the fixed template. Primers can be reversibly blocked to prevent or inhibit extension. Single-stranded templates can be sequenced using primers hybridized at the 3' end. The sequencing primer can be removed after sequencing, and the fixed primer is unblocked to release the extendable 3' hydroxyl. These primers can be used to replicate the template using a bridged chain resynthesis to produce a second fixed template complementary to the first template. Removing the first template from the surface allows the new single-stranded second template to be sequenced from the 3' end as well. Therefore, the two ends of the original immobilized template can be sequenced. This technology allows paired-end reading, where the template is amplified using a single extendable fixed primer, for example as described in the Polony technology (Nucleic Acids Research 27, 24, e34 (1999)) or emulsion PCR (Science 309, 5741, 1728-1732 (2005); Nature 437, 376-380 (2005)).

[0089] As provided herein, in a non-limiting example of the present disclosure, changing the energy recycling system used in an amplification system for sequencing enables better regulation of the kinetics of the amplification reaction used, thereby allowing for higher monoclonality to be obtained in the cluster, compared to energy recycling systems previously used in amplification (e.g., creatine kinase / phosphocreatine systems), which in turn leads to higher quality sequencing results, and thereby increases the throughput of sequencing results.

[0090] like Figure 6 As shown in , the inventors have discovered that the generation of ATP during amplification by polyphosphate kinase provides a source of ATP used by the recombinase for homology searching and strand invasion.

[0091] The inventors have also found that removing inorganic pyrophosphate from the amplification composition, such as by adding polyphosphate kinase, has many advantages in nucleic acid amplification and sequencing methods. Specifically, the inventors have found that the removal of inorganic pyrophosphate can be used to improve clustering kinetics, and then reduce clustering time (and therefore reduce turnover time) and / or increase signal intensity (and therefore increase sequence signal-to-noise ratio).

[0092] Specifically, the inventors have discovered that improving clustering dynamics by removing or reducing PPi leads to improvements in sequencing performance, including but not limited to increases in intensity, %PF, Q30, and yield (g). "%PF" refers to the % of reads that pass the purity filter (purity is the ratio of the intensity of the brightest base divided by the sum of the intensity of the brightest base and the intensity of the second brightest base). "Q30" refers to the percentage of bases with a quality score of 30 or higher. The quality score is "an estimate of the probability that a base is miscalled: q = -10×log10(p)". "Yield" refers to the number of bases produced in a run. This is shown in Figure 5. Here, adding inorganic pyrophosphatase as a reference means reducing to PPi (such as Figure 5E ), increasing strength at 0.3U and 1.2U ( Figure 5A )、%PF( Figure 5B )、Q30( Figure 5C ) and yield (g)( Figure 5D ).

[0093] Additionally, increasing clustering intensity also allows amplification / clustering to occur in smaller wells, where a decrease in well size requires an increase in signal intensity.

[0094] In addition, as mentioned above, the accumulation of inorganic pyrophosphate causes DNA polymerase to stagnate. When DNA polymerase encounters structured secondary features (such as G-quadruplexes), this is problematic, causing parts of the library to not be clustered / amplified, and therefore not sequenced. The removal of inorganic pyrophosphate prevents or inhibits the stagnation of DNA polymerase, and therefore reduces sequence-specific errors, because the polymerase is able to cluster / amplify the structured regions of the genome.

[0095] Finally, in addition to improving clustering dynamics (e.g., clustering time and signal intensity), adding polyphosphate kinase can also significantly reduce the amount of required clustering / amplification reagents by up to 50%. As mentioned, in amplification or clustering reactions, it may be necessary to add the amplification composition more than once (the number of times the amplification composition is added to the circulation pool can be referred to as "pushing"). Multiple pushes may be necessary to achieve the desired level of sequence signal intensity. The removal of inorganic pyrophosphate can significantly increase the sequence signal intensity with a single push. Therefore, by reducing the PPi level, it is possible to additionally halve the amount of the required amplification composition (i.e., halve COG (cost of goods)) without affecting clustering / amplification intensity.

[0096] As used herein, the term "cluster" may refer to a clonal group of template polynucleotides (e.g., DNA or RNA) bound in a single hole of a flow cell."Cluster" may contain a sufficient number of copies of a single template polynucleotide so that the cluster can output a signal (e.g., optical signal) that allows a single sequencing read of the cluster. "Cluster" may include, for example, about 500 to about 2000 copies, preferably about 600 to about 1800 copies, more preferably about 700 to about 1600 copies, even more preferably about 800 to 1400 copies, also even more preferably about 900 to 1200 copies, most preferably about 1000 copies of a single template polynucleotide. The copy of a single template polynucleotide may include at least about 50% of all polynucleotides in a single hole of a flow cell, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, also even more preferably at least about 90%, most preferably about 95%, 98%, 99% or 100%, thereby providing a substantially monoclonal "cluster".

[0097] In one embodiment, examples of the present disclosure relate to an amplification composition comprising an inorganic polyphosphate and a polyphosphate kinase.

[0098] As used herein, the term "inorganic polyphosphate" may refer to a system of two or more phosphoric acid residues connected by a phosphoric anhydride bond. The system may be linear.

[0099] The inorganic polyphosphate may be present in acid form, salt form, or a combination thereof. In the case where the inorganic polyphosphate is present in salt form, the inorganic polyphosphate may contain cations (excluding H+ ). For example, the cation may be selected from "metal cation" or "non-metal cation". The metal cation may include an alkali metal ion (e.g., lithium, sodium, potassium, rubidium or cesium ion). The non-metal cation may include an ammonium salt (e.g., an alkylammonium salt) or a phosphonium salt (e.g., an alkylphosphonium salt).

[0100] Inorganic polyphosphates are soluble in aqueous media.

[0101] As used herein, the term "polyphosphate kinase" may refer to an enzyme that catalyzes the following reaction:

[0102] PolyP n +ADP→PolyP n-1 +ATP

[0103] Among them, PolyP n refers to an inorganic polyphosphate with "n" phosphate residues, ADP refers to adenosine diphosphate, PolyP n-1 refers to an inorganic polyphosphate having "n-1" phosphate residues, and ATP refers to adenosine triphosphate.

[0104] The composition may include an inorganic polyphosphate at a concentration of about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 2 μM to about 10 μM. Alternatively, the inorganic polyphosphate is present in a weight % relative to the total weight % of the composition by dry mass of about 0.01 wt % to about 5.0 wt %, about 0.02 wt % to about 4.5 wt %, about 0.05 wt % to about 4.0 wt %, about 0.08 wt % to about 3.5 wt %, about 0.1 wt % to about 3.0 wt %, about 0.2 wt % to about 2.5 wt %, or about 0.5 wt % to about 2.0 wt %.

[0105] The composition may comprise a polyphosphate kinase at a concentration of about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 2 μM to about 10 μM. Alternatively, the composition comprises a polyphosphate kinase between about 0.01 U / μL and about 100 U / μL, between about 0.1 U / μL and about 50 U / μL, between about 0.2 U / μL and about 30 U / μL, between about 0.3 U / μL and about 20 U / μL, between about 0.5 U / μL and about 10 U / μL, or between about 1.0 U / μL and about 5.0 U / μL. For example, the composition may comprise about 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, 0.7 U / μL, 0.8 U / μL, 0.9 U / μL, 1.0 U / μL, 1.1 U / μL, 1.2 U / μL, 1.3 U / μL, 1.4 U / μL, 1.5 U / μL, 1.6 U / μL, 1.7 U / μL, 1.8 U / μL, 1.9 U / μL, or about 2.0 U / μL of polyphosphate kinase. Alternatively, the polyphosphate kinase is present in a weight % relative to the total weight % of the composition on a dry mass basis from about 0.01 wt % to about 5.0 wt %, from about 0.02 wt % to about 4.5 wt %, from about 0.05 wt % to about 4.0 wt %, from about 0.08 wt % to about 3.5 wt %, from about 0.1 wt % to about 3.0 wt %, from about 0.2 wt % to about 2.5 wt % or from about 0.5 wt % to about 2.0 wt %.

[0106] Inorganic polyphosphate can comprise the first inorganic polyphosphate with less than 50 phosphate residues.For example, the first inorganic polyphosphate can comprise 2 to 50 phosphate residues, preferably 5 to 45 phosphate residues, more preferably 10 to 40 phosphate residues, even more preferably 15 to 35 phosphate residues.In a preferred embodiment, the first inorganic polyphosphate can be pyrophosphate (two phosphate residues).The number of phosphate residues can refer to the average number of phosphate residues (for example, the median number of phosphate residues).

[0107] The inorganic polyphosphate may include a second inorganic polyphosphate having more than 100 phosphate residues. For example, the second inorganic polyphosphate may contain 100 to 10,000 phosphate residues, preferably 150 to 5,000 phosphate residues, more preferably 200 to 2,000 phosphate residues, and even more preferably 250 to 1,000 phosphate residues. The number of phosphate residues may refer to the average number of phosphate residues (e.g., the median number of phosphate residues).

[0108] The ratio of the first inorganic polyphosphate to the second inorganic polyphosphate can be from about 99:1 to about 1:99, from about 98:2 to about 2:98, from about 95:5 to about 5:95, from about 90:10 to about 10:90, from about 85:15 to about 15:85, from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 50:50. In some embodiments (e.g., for a first polyphosphate composition as described herein), the ratio of the first inorganic polyphosphate to the second inorganic polyphosphate is from about 99:1 to about 50:50, preferably from about 98:2 to about 55:45, more preferably from about 95:5 to about 60:40, even more preferably from about 90:10 to about 65:35, still even more preferably from about 85:15 to about 70:30, and most preferably from about 80:20 to about 75:25. In some embodiments (e.g., for a second polyphosphate composition as described herein), the ratio of the first inorganic polyphosphate to the second inorganic polyphosphate is from about 50:50 to about 1:99, preferably from about 45:55 to about 2:98, more preferably from about 40:60 to about 5:95, even more preferably from about 35:65 to about 10:90, still even more preferably from about 30:70 to about 15:85, and most preferably from about 25:75 to about 20:80.

[0109] The polyphosphate kinase may be selected from the group consisting of: a polyphosphate kinase of the PPK1 family, a polyphosphate kinase of the PPK2 family, and a polyphosphate kinase of the PPK3 family. Preferably, the polyphosphate kinase is selected from the group consisting of: a polyphosphate kinase of the PPK1 family, and a polyphosphate kinase of the PPK2 family.

[0110] The polyphosphate kinase may be a thermophilic or a mesophilic polyphosphate kinase.

[0111] In one embodiment, the polyphosphate kinase is derived from a thermophilic bacterium (including a hyperthermophile). Examples of thermophiles or hyperthermophiles include microorganisms from the Thermococcaceae, Thermaceae, or Thermotogaceae families; or from the genus Thermus, Meiothermus, Thermococcus, Pyrococcus, or Thermotoga. In one embodiment, the thermophile can be selected from the group consisting of Thermococcus kodacaraensis, Meiothermus ruber, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus species GB-D, Pyrococcus woesei, Thermus aquaticus, Thermus brokianus, Thermus caldophilus, Thermus filiformis, Thermus flavus, Thermococcus fumiculans, Thermococcus gorgonarius, Thermococcus litoralis, Thermotoga maritima, Thermotoga neoapollo. neopolitana and Thermus thermophilus.

[0112] In one embodiment, the thermophile is from the genus Thermus. In one embodiment, the thermophile is Thermus thermophilus, and the polyphosphate kinase may comprise the following sequence selected from the group consisting of:

[0113] HLLPEASWLQFNRRVLLQTERPDFPLLERLRFLGIWNRNLDEFFAARIAKPFLKSRRGPDHLALLQEALDQAKLARARYQNLLQEAFPRLRVLDPGELDDLDWLYFRVFLAEEVAPKTDLIPWEAAQDLSHSALYFASERYLVRLPQDLPR LVEVPGREGTYVRLGALMRWRSDLLLPEEAPLYEFRVLRLLESERVRADWNELAESLEGRQEGTPTLLVVEEGFPEAWLDALRRALGLFLEEVFALKPPLNLSLVDTLVAQGPPEWRFPPFRPERPRTFLKNPLALLGKRDVLLYHPFEDY AAVERFAEAALAEEVEEVWATLYRTGEENPLAEALIAAARKGKRVHVLLEGRARFDELLNLRWYLRLVRAGVEVLPLPERKVHAKAFLILTREGGYAHLGTGNYNPTNGHHYTDFSLFTARKEVVAEVRAFFQAMAEEKTPRLGLLRTGEG IRRLLLEAVLHEAHPKGRLILKFNHLTDPELLEALVYAASRGARVDLLVRSTLTRLHPAIRAKSLVGRFLEHARAAAFRAGGEWRVYLTSADAMPRNFQNRFELLFPVLDKEAKKKVLKVLKRQVRDDRNSFLLTPEGEKRLWGGRHDAQRL

[0114] (SEQ ID NO: 1).

[0115] In one embodiment, the thermophile is from the genus Thermus. In one embodiment, the thermophile is Thermus rubrum, and the polyphosphate kinase may comprise the following sequence or a functional variant or functional fragment thereof:

[0116] KKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLVVRVK NLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE

[0117] (SEQ ID NO:2).

[0118] In one embodiment, the polyphosphate kinase is derived from a mesophilic bacterium. Examples of mesophilic bacteria include Saccharomyces cerevisiae and Escherichia coli. In one embodiment, the polyphosphate kinase comprises a sequence as shown in SEQ ID NO: 3 or a functional variant or functional fragment thereof:

[0119] MGQEKLYIES EIPSDKVPRFVNLPPEAPRRRKPMILLDNILRYCLDDIFKGFFDYDALNAYSMKMTRDAEYDLVHEMEASLMELMSSSLKQRLTAEPVRFVYQRDMPNALVEVLREKLTISRYDSIVPGGRYHNFKDFINFPNVGKANLVNKPLPRLRHIWFDKAQFRNGFDAIRERDVLLY YPYHTFEHVLELLRQASFDPSVLAIKINIYRVAKDSRIIDSMIHAAHNGKKVTVVVELQARFDEEANIHWAKRLTEAAGVHVIFSAPGLKIHAKLFLISRKENGEVVRYAHIGTGNFNEKTARLYTDYSLLTADARITNEVRRVFNFIENPYRPVTFDYLMVSPQNSRRLLYE MVDREIANAQQGLPSGITLKLNNLVDKGLVDRLYAASSSGVPVNLLVRGMCSLIPNLEGISDNIRAISIVDRYLEHDRVYIFENGGDKKVYLSSADWMTRNIDYRIEVATPLLDPRLKQRVLDIIDILFSDTVKARYIDKELSNRYVPRGNRRKVRAQLAIYDYIKSLEQPE

[0120] (SEQ ID NO:3).

[0121] As used herein, the term "thermophilic" or "thermostable" may refer to a protein that is not substantially denatured at high temperatures, for example, above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, above 80°C, above 85°C, above 90°C, above 95°C, or above 100°C.

[0122] The polyphosphate kinase may have an optimal operating temperature of about 50°C to about 75°C, preferably about 55°C to about 70°C, or more preferably about 60°C to about 65°C; for example, the polyphosphate kinase may have an optimal operating temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C or about 75°C.

[0123] As used herein, the term "optimal operating temperature" may refer to a temperature at which the catalytic activity of an enzyme reaches a peak maximum.

[0124] As used herein, the term "functional variant" refers to a variant polypeptide sequence or portion of a polypeptide sequence that retains the biological function of the complete non-variant sequence. For example, a functional variant of a polyphosphate kinase is capable of catalyzing the conversion of inorganic polyphosphate and ADP into ATP.

[0125] Functional variants also include variants of polypeptides of interest, which have sequence changes that do not affect function, such as in non-conservative residues. Also include substantially identical variants, that is, compared with the wild-type sequence shown herein, only have some sequence variations, such as in non-conservative residues, and have biological activity. The changes of polypeptide sequences that do not affect the functional properties of polypeptides are well known in the art. For example, amino acid alanine (a hydrophobic amino acid) can be replaced by another hydrophobic residue (such as glycine) or a hydrophobic residue (such as valine, leucine or isoleucine) with lower hydrophobicity. Similarly, the change that causes a negatively charged residue to be replaced by another (such as aspartic acid is replaced by glutamic acid) or a positively charged residue to be replaced by another (such as lysine is replaced by arginine) can also be expected to produce functionally equivalent products. Each modification in the proposed modification is within the scope of routine techniques in the art, such as determining the retention of the biological activity of the encoded product.

[0126] As used in any aspect described herein, a "functional variant" has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, %. The invention relates to a polypeptide having an overall sequence identity of at least 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, For example, Emboss Stretcher from EMBL-EBI can be used: https: / / www.ebi.ac.uk / Tools / psa / emboss_stretcher / (using default parameters: paired output format for proteins, Matrix=BLOSUM62, Gap open=1, Gap extend=1; paired output format for nucleotides, Matrix=DNAfull, Gap open=16, Gap extend=4).

[0127] As used herein, the term "functional fragment" refers to a functionally active series of consecutive amino acids from a longer polypeptide or protein. For example, a functional fragment may retain the catalytic activity of a polyphosphate kinase as described above.

[0128] The composition may further comprise a recombinase. The recombinase may be a thermophilic recombinase.

[0129] As used herein, the term "recombinase" may refer to an enzyme that promotes the invasion of target nucleic acid by polymerase and the extension of the primer carried out by the polymerase, and the polymerase uses the target nucleic acid as the template formed by the amplicon. The process can be repeated as a chain reaction, wherein the amplicon produced by each round of invasion / extension is used as the template in the subsequent round. Since no denaturation cycle (for example, via heating or chemical denaturation) is required, the process can occur more rapidly than standard PCR. Therefore, the amplification promoted by the recombinase can be carried out isothermally. It is usually desirable to include ATP or other nucleotides (or its non-hydrolyzable analogs in some cases) in the amplification reagent promoted by the recombinase to promote amplification. The mixture of recombinase and single-stranded binding (SSB) protein is particularly useful because SSB can further promote amplification. Recombinase may include, for example, RecA protein, T4 uvsX protein, any homologous protein from any gate or protein complex or its functional variant. Eukaryotic RecA homologues are usually named Rad51 after the first member to be identified in the group. Other non-homologous recombinases may be used instead of RecA, such as RecT or RecO.

[0130] In some preferred embodiments, the recombinase may be UvsX. In one embodiment, UvsX comprises or consists of SEQ ID NO: 8 or 9 or a functional fragment or functional variant thereof.

[0131] In other preferred embodiments, the recombinase may be thermophilic UvsX. In one embodiment, thermophilic UvsX comprises or consists of SEQ ID NO: 10 or 11 or a functional fragment or functional variant thereof.

[0132] The composition may further comprise a single-stranded nucleotide binding protein.

[0133] As used herein, the term "single-stranded nucleotide binding protein" may refer to any protein that has the function of binding to a single-stranded nucleic acid, for example, to prevent or inhibit premature annealing, to protect the single-stranded nucleic acid from being digested by nucleases, to remove secondary structures from the nucleic acid, or to promote the replication of the nucleic acid. The term is intended to include, but is not necessarily limited to, proteins formally identified as single-stranded binding proteins by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Exemplary single-stranded binding proteins include, but are not limited to, E. coli SSB, T4 gp32, T7 gene 2.5SSB, bacteriophage phi 29SSB, any homologous protein or protein complex from any gate, or functionalized variants thereof.

[0134] The composition may also include a polymerase. Preferably, the polymerase may be a strand displacement polymerase. In some preferred embodiments, the polymerase may be a DNA polymerase. In other preferred embodiments, the polymerase may be an RNA polymerase. The polymerase may be a thermophilic polymerase.

[0135] As used herein, the term "polymerase" may refer to an enzyme that uses nucleic acid as a template strand to produce complementary replication of a nucleic acid molecule. Typically, a DNA polymerase is bound to a template strand and then moves downward along the template strand, adding nucleotides to the free hydroxyl groups at the 3' end of the growing nucleic acid strand in sequence. DNA polymerases typically synthesize complementary DNA molecules from DNA templates and RNA polymerases typically synthesize RNA molecules (transcription) from DNA templates. Polymerases can use short RNA or DNA strands (referred to as primers) to start chain growth. Some polymerases can displace the strands upstream of the site where they add bases to the strand. Such polymerases are referred to as strand displacements, meaning that they have the activity of removing complementary strands from the template strands read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, large fragments of Bst (Bacillus stearothermophilus (Bacillus stearothermophilus)) polymerases, exo-Klenow polymerases, or sequencing-grade T7 exo-polymerases. Some polymerases degrade the strands in front of them, thereby effectively displacing the front strands (5' exonuclease activity) with the strands growing behind. Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0136] Said composition may also include nucleotide triphosphates (NTPs). Preferably, nucleotide triphosphates may be deoxynucleotide triphosphates (dNTPs). More preferably, said composition includes a variety of NTPs or dNTPs, and preferably includes a mixture, for example, includes a variety of dATP, dGTP, dCTP and dTTP (for DNA clustering / synthesis) or ATP, GTP, CTP and UTP (for RNA clustering / synthesis). In one embodiment, the concentration of dNTPs may be between 0.1mM and 2mM, preferably between 0.2mM and 1.5mM, more preferably between 0.3mM and 1.2mM, even more preferably between 0.3mM and 0.6mM; for example, concentration may be selected from 0.3mM, 0.6mM and 1.2mM.

[0137] As used herein, the term "nucleotide triphosphate" may refer to a molecule containing a nitrogenous base (e.g., adenine, thymine, cytosine, guanine, uracil) bound to a 5-carbon sugar (e.g., ribose, or deoxyribose), wherein three phosphate groups are bound to the sugar.

[0138] As used herein, the term "deoxynucleotide triphosphate" or (dNTP) may refer to a molecule containing a nitrogenous base (e.g., adenine, thymine, cytosine, guanine, uracil) bound to a deoxyribose sugar, wherein three phosphate groups are bound to the deoxyribose sugar.

[0139] In some embodiments, the composition may not include creatine kinase and / or creatine phosphate.

[0140] The composition may comprise an inorganic polyphosphate, a polyphosphate kinase, and at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, and nucleotide triphosphates (NTPs). Preferably, the composition may comprise an inorganic polyphosphate, a polyphosphate kinase, and at least two selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, and nucleotide triphosphates (NTPs). More preferably, the composition may comprise an inorganic polyphosphate, a polyphosphate kinase, and at least three selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, and nucleotide triphosphates (NTPs).

[0141] Preferably, the composition further comprises at least one selected from the group consisting of: a recombinase, NTP and a single-stranded nucleotide binding (SSB) protein. More preferably, the composition further comprises at least two selected from the group consisting of: a recombinase, NTP and a single-stranded nucleotide binding (SSB) protein.

[0142] Preferably, the composition may further comprise a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein.

[0143] Preferably, the composition may comprise an inorganic polyphosphate, a polyphosphate kinase, a recombinase, a single-stranded nucleotide binding protein, a polymerase and nucleotide triphosphates (NTPs).

[0144] In some embodiments, the composition may not include one or more primers, i.e., amplification primers or sequencing primers. Therefore, the composition may not include primers. That is, the composition may not include any nucleic acid sequence that can initiate DNA synthesis (by polymerase). Primers can be free nucleic acid sequences of base pairs between 18 and 22, more preferably base pairs between 15 and 30. The GC content of the free nucleic acid sequence can also be between 50% and 55%, and preferably, GC-locks (G or C in the last 5 bases of the sequence) can be provided at the 3' end. The melting temperature of the primer can be between 40°C and 60°C, more preferably between 50°C and 55°C. Primers can also be complementary or substantially complementary (having, for example, at least 80% overall sequence identity) to the target sequence or its complement that is expected to be clustered with the composition. Primers can also include one or more restriction sites.

[0145] In some embodiments, the composition may further comprise a nucleic acid template. The nucleic acid template may further comprise an adapter sequence as described herein, wherein preferably, the adapter sequence comprises at least one of P5, P5', P7 and P7', the sequences of which are described below.

[0146] The composition may not contain PEG.

[0147] The composition may comprise a buffer.Preferably, the amplification composition is buffered to a pH of about 6.0 to about 9.0, preferably about 6.5 to about 8.8, more preferably about 7.5 to about 8.7, even more preferably about 8.3 to about 8.6.

[0148] Said composition can be provided in dried form (e.g., freeze-dried form). In this case, before being used for amplification, the amplification composition can be rehydrated, for example, with water or a buffer solution. In other embodiments, the amplification composition can be provided as a solution (e.g., as the aqueous solution).

[0149] The composition may also include an excipient. Suitable excipients may include surfactants, such as anionic surfactants, including alkyl sulfates (e.g., ammonium lauryl sulfate, sodium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, sodium docusate, docusate), alkyl sulfonates (e.g., perfluorooctane sulfonate, perfluorobutane sulfonate), alkyl phosphates (e.g., alkyl-aryl ether phosphates, alkyl ether phosphates) and alkyl carboxylates (e.g., sodium stearate, sodium lauroyl sarcosinate, perfluorononanoate, perfluorooctanoate); cationic surfactants, including quaternary ammonium salts (e.g., cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, dimethyldioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide); nonionic surfactants, including fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid ethoxylates, ethoxylated amines or fatty acid amides, poloxamer, polysorbates (e.g., polyethylene glycol sorbitan alkyl esters (Tween)). Other excipients may include enzyme stabilizers such as dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) and 2-mercaptoethanol (BME). Still further excipients may include molecular crowding agents such as polyethylene glycol (PEG), dextran, and epichlorohydrin-sucrose polymers (eg, Ficoll); in some embodiments, PEG may be excluded.

[0150] Preferably, the amplification composition may be a clustered composition.

[0151] The term "amplification composition" refers to a composition suitable for amplification of a target nucleic acid template.

[0152] In contrast, a "clustered composition" refers to a composition suitable for amplifying a (single) target sequence into clusters as described above (i.e., the composition is suitable for cluster generation, particularly for generating monoclonal clusters), not only for any amplification method. In one embodiment, the composition is also not suitable for detection or sequencing of nucleic acid templates. For example, in one embodiment, the composition does not include fluorescent entities, such as probes, nucleotides labeled with fluorescent entities, and / or primers labeled with fluorescent entities. Alternatively, the composition does not include leuco dyes / reagents labeled with leuco dyes.

[0153] In one embodiment, the composition can be a resynthesis composition. Resynthesis refers to the step between the first sequencing read and the second sequencing read, where the template is resynthesized using a bridge strand to produce a second fixed template that is complementary to the first fixed template. Thus, the same composition as described herein can be used for resynthesis.

[0154] In one embodiment, the composition can be a sequencing-by-synthesis amplification composition.

[0155] In another embodiment, examples of the present disclosure relate to a kit comprising an inorganic polyphosphate and a polyphosphate kinase. The polyphosphate kinase can be provided separately from the inorganic polyphosphate. For example, the polyphosphate kinase can be in a different container from the inorganic polyphosphate.

[0156] Preferably, the kit may comprise an amplification composition as described herein.

[0157] The kit may also include a recombinase as described herein. The recombinase may be provided separately from the inorganic polyphosphate and / or polyphosphate kinase (e.g., separate from the amplification composition as described herein). For example, the recombinase may be in a container different from the inorganic polyphosphate and / or polyphosphate kinase (e.g., a container different from the amplification composition as described herein).

[0158] The test kit may also include a single-stranded nucleotide binding protein as described herein. The single-stranded nucleotide binding protein may be provided separately from an inorganic polyphosphate and / or a polyphosphate kinase (e.g., separate from an amplification composition as described herein). For example, the single-stranded nucleotide binding protein may be in a container different from an inorganic polyphosphate and / or a polyphosphate kinase (e.g., a container different from an amplification composition as described herein).

[0159] The kit may also include a polymerase as described herein. The polymerase may be provided separately from the inorganic polyphosphate and / or polyphosphate kinase (e.g., separate from the amplification compositions described herein). For example, the polymerase may be in a container different from the inorganic polyphosphate and / or polyphosphate kinase (e.g., a container different from the amplification compositions described herein).

[0160] The test kit may also include a variety of nucleotide triphosphates (NTPs) and mixtures as described herein. Nucleotide triphosphates may be provided separately from inorganic polyphosphates and / or polyphosphate kinases (e.g., separate from amplification compositions as described herein). For example, nucleotide triphosphates may be in a container different from inorganic polyphosphates and / or polyphosphate kinases (e.g., a container different from amplification compositions as described herein).

[0161] The kit may include inorganic polyphosphate, polyphosphate kinase and at least one selected from the group consisting of the following items: recombinase, single-stranded nucleotide binding protein, polymerase and nucleotide triphosphate (NTP). Preferably, the kit may include inorganic polyphosphate, polyphosphate kinase and at least two selected from the group consisting of the following items: recombinase, single-stranded nucleotide binding protein, polymerase and nucleotide triphosphate (NTP). More preferably, the kit may include inorganic polyphosphate, polyphosphate kinase and at least three selected from the group consisting of the following items: recombinase, single-stranded nucleotide binding protein, polymerase and nucleotide triphosphate (NTP). One or more (e.g., each of these components) can be provided separately from inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from amplification compositions as described herein). For example, one or more (e.g., each of these components) can be in a container different from inorganic polyphosphate and / or polyphosphate kinase (e.g., in a container different from amplification compositions as described herein).

[0162] Preferably, the kit also comprises at least one selected from the group consisting of the following items: recombinase, NTP and single-stranded nucleotide binding (SSB) protein. More preferably, the composition also comprises at least two selected from the group consisting of the following items: recombinase, NTP and single-stranded nucleotide binding (SSB) protein. One or more (e.g., each of these components) can be provided separately from inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from amplification compositions as described herein). For example, one or more (e.g., each of these components) can be in a container different from inorganic polyphosphate and / or polyphosphate kinase (e.g., in a container different from amplification compositions as described herein).

[0163] Preferably, the kit may include inorganic polyphosphate, polyphosphate kinase, recombinase, NTP and single-stranded nucleotide binding (SSB) protein. One or more (e.g., each of these components) can be provided separately from inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from amplification compositions as described herein). For example, one or more (e.g., each of these components) can be in a container different from inorganic polyphosphate and / or polyphosphate kinase (e.g., in a container different from amplification compositions as described herein).

[0164] Preferably, the kit may include inorganic polyphosphate, polyphosphate kinase, recombinase, single-stranded nucleotide binding protein, polymerase and nucleotide triphosphate (NTP). One or more (e.g., each of these components) can be provided separately from inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from amplification compositions as described herein). For example, one or more (e.g., each of these components) can be in a container different from inorganic polyphosphate and / or polyphosphate kinase (e.g., in a container different from amplification compositions as described herein).

[0165] The kit may also include an excipient as described herein. The excipient may be provided separately from the inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from the amplification composition as described herein). For example, the excipient may be in a container different from the inorganic polyphosphate and / or polyphosphate kinase (e.g., in a container different from the amplification composition as described herein).

[0166] The kit may also include one or more reagents for preparing template nucleic acid sequences for clustering and sequencing (i.e., library preparation reagents). In one embodiment, the kit may also include adapter sequences. Adapter sequences can be configured so that they can be connected to the nucleic acid template to be sequenced. In some preferred embodiments, the kit may include a first adapter sequence, which includes a sequence according to SEQ ID NO.4 (P5) or a variant or fragment thereof. In other preferred embodiments, the kit may include a second adapter sequence, which includes a sequence according to SEQ ID NO.5 (P7) or a variant or fragment thereof. In other preferred embodiments, the kit may include a third adapter sequence, which includes a sequence according to SEQ ID NO.6 (P5') or a variant or fragment thereof. In other preferred embodiments, the kit may include a fourth adapter sequence, which includes a sequence according to SEQ ID NO.7 (P7') or a variant or fragment thereof. More preferably, the kit may include at least two selected from the group consisting of the following items: a first adapter sequence, a second adapter sequence, a third adapter sequence, and a fourth adapter sequence. Even more preferably, the kit may comprise at least three selected from the group consisting of: a first adapter sequence, a second adapter sequence, a third adapter sequence, and a fourth adapter sequence. Still even more preferably, the kit may comprise a first adapter sequence, a second adapter sequence, a third adapter sequence, and a fourth adapter sequence. The adapter sequence (e.g., each of the adapter sequences) can be provided separately from the inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from the amplification composition described herein). For example, the adapter sequence (e.g., each of the adapter sequences) can be in a different container from the inorganic polyphosphate and / or polyphosphate kinase (e.g., in a different container from the amplification composition described herein). The kit may also comprise a metal cofactor composition. The metal cofactor can be configured to activate one or more enzymes in the amplification composition. For example, the metal cofactor can be configured to activate a recombinase and / or a polymerase. Preferably, the metal cofactor composition comprises magnesium ions (e.g., magnesium acetate, magnesium chloride). The metal cofactor composition can be provided separately from the inorganic polyphosphate and / or polyphosphate kinase (e.g., provided separately from an amplification composition as described herein). For example, the metal cofactor composition can be in a different container than the inorganic polyphosphate and / or polyphosphate kinase (e.g., in a different container than an amplification composition as described herein).

[0167] The kit may further comprise a solid support, preferably a flow cell. Preferably, the moss primers (P5 and P7) are immobilized on the flow cell as described in detail above.

[0168] In another embodiment, embodiments of the present disclosure relate to the use of an amplification composition as described herein or a kit as described herein in amplifying a nucleic acid template or in sequencing a nucleic acid sequence.

[0169] In another embodiment, embodiments of the present disclosure are directed to a method of amplifying a nucleic acid template, wherein the method comprises recycling ADP to ATP using an inorganic polyphosphate and a polyphosphate kinase.

[0170] The method of amplifying a nucleic acid template may include adding an amplification composition as described herein. The composition may be added to a sample containing a nucleic acid template to be amplified. In particular, "addition" may refer to adding the composition to a flow cell before, after, or simultaneously with a sample containing a nucleic acid template. The nucleic acid template may contain an adapter sequence as described above (comprising at least one of P5, P5', P7, and P7').

[0171] The method of amplifying a nucleic acid template may include adding a first polyphosphate composition and a second polyphosphate composition, wherein in the first polyphosphate composition, the amount of the first inorganic polyphosphate is higher relative to the amount of the second inorganic polyphosphate, and wherein in the second polyphosphate composition, the amount of the second inorganic polyphosphate is higher relative to the amount of the first inorganic polyphosphate.

[0172] The first polyphosphate composition may comprise a first inorganic polyphosphate as defined herein and a second inorganic polyphosphate as defined herein, wherein the ratio of the first inorganic polyphosphate to the second inorganic polyphosphate is from about 99:1 to about 50:50, preferably from about 98:2 to about 55:45, more preferably from about 95:5 to about 60:40, even more preferably from about 90:10 to about 65:35, still even more preferably from about 85:15 to about 70:30, and most preferably from about 80:20 to about 75:25.

[0173] The second polyphosphate composition may comprise a first inorganic polyphosphate as defined herein and a second inorganic polyphosphate as defined herein, wherein the ratio of the first inorganic polyphosphate to the second inorganic polyphosphate is from about 50:50 to about 1:99, preferably from about 45:55 to about 2:98, more preferably from about 40:60 to about 5:95, even more preferably from about 35:65 to about 10:90, still even more preferably from about 30:70 to about 15:85, and most preferably from about 25:75 to about 20:80.

[0174] Preferably, the first polyphosphate composition is added before the second polyphosphate composition.

[0175] Amplification can be performed by exclusive amplification. Amplification can be performed by bridging amplification.

[0176] Amplification may be performed at a temperature of about 50° C. to about 75° C., about 55° C. to about 70° C., or about 60° C. to about 65° C. Preferably, amplification is performed isothermally.

[0177] In another embodiment, an embodiment of the present disclosure relates to a method for sequencing a nucleic acid sequence, wherein the method comprises amplifying a nucleic acid template as described herein; and sequencing the amplified nucleic acid template.

[0178] The step of sequencing the amplified nucleic acid template may include performing a single read. In other embodiments, the step of sequencing the amplified nucleic acid template includes performing a paired end read.

[0179] The step of sequencing the amplified nucleic acid template may include performing a first sequencing read and a second sequencing read.

[0180] The step of sequencing the amplified nucleic acid template can be performed using sequencing by synthesis technology or sequencing by ligation technology.

[0181] The method of sequencing a nucleic acid sequence can be performed isothermally.

[0182] One or more steps in the method of sequencing a nucleic acid sequence are performed at a temperature of about 50° C. to about 75° C., about 55° C. to about 70° C., or about 60° C. to about 65° C. Preferably, all steps in the method of sequencing a nucleic acid sequence are performed at a temperature of about 50° C. to about 75° C., about 55° C. to about 70° C., or about 60° C. to about 65° C.

[0183] When bridging amplification is used, the step of sequencing the amplified nucleic acid template may include a first linearization step. The first linearization step may be performed after (eg, immediately after) the step of amplifying the nucleic acid template.

[0184] The step of sequencing the amplified nucleic acid template may include the step of adding an exonuclease. The step of adding an exonuclease may be performed after the step of amplifying the nucleic acid template. For example, the step of adding an exonuclease may be performed after (e.g., immediately after) the first linearization step.

[0185] Preferably, the exonuclease is a thermophilic exonuclease. More preferably, the exonuclease is derived from a thermophilic organism, such as Pyrococcus furiosus.

[0186] Preferably, the exonuclease has an optimal operating temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

[0187] The step of sequencing the amplified nucleic acid template may include a first step of dehybridizing the complementary strand bound to the nucleic acid template with a dehybridizing agent. The dehybridizing agent may be configured to disengage the complementary strand from the nucleic acid template, thereby allowing the complementary strand to be washed off. The first step of dehybridizing the complementary strand may be performed after the step of amplifying the nucleic acid template. For example, the first step of dehybridizing the complementary strand may be performed after (e.g., immediately after) the step of adding an exonuclease.

[0188] The step of sequencing the amplified nucleic acid template may include a first step of hybridizing a sequencing primer to the nucleic acid template. The first step of hybridizing the sequencing primer may be performed after the step of amplifying the nucleic acid template. For example, the first step of hybridizing the sequencing primer may be performed after (e.g., immediately after) the first step of dehybridizing the complementary strand.

[0189] The step of sequencing the amplified nucleic acid template may include a first step of performing sequencing by synthesis. The first step of performing sequencing by synthesis may be performed after the step of amplifying the nucleic acid template. For example, the first step of performing sequencing by synthesis may be performed after the first step of hybridizing the sequencing primer (e.g., immediately thereafter).

[0190] In the case of performing a second sequencing read (e.g., for paired end reading), the step of sequencing the amplified nucleic acid may also include a step of removing a blocking group from a hydroxyl group of a primer (e.g., a P5 or P7 primer). For example, the step of removing the blocking group may include removing the phosphate group using a blocking group phosphatase. The step of removing the blocking group may be performed after the step of amplifying the nucleic acid template. For example, the step of removing the blocking group may be performed after (e.g., immediately after) the first step of sequencing by synthesis.

[0191] Preferably, the blocking group phosphatase is a thermophilic phosphatase. More preferably, the blocking group phosphatase is derived from a thermophilic organism, such as Pyrococcus furiosus.

[0192] Preferably, the phosphatase has an optimal operating temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

[0193] Where a second sequencing read is performed (e.g., for paired end reading), the step of sequencing the amplified nucleic acid may further include the step of generating a complementary form of the amplified nucleic acid template. The step of generating a complementary form of the amplified nucleic acid template may involve using an amplification method as described herein, e.g., using an inorganic polyphosphate and a polyphosphate kinase. The step of generating a complementary form of the amplified nucleic acid template may be performed after the step of amplifying the nucleic acid template. For example, the step of generating a complementary form of the amplified nucleic acid template may be performed after (e.g., immediately after) the step of removing the blocking group.

[0194] Where a second sequencing read is performed (e.g., for paired end reading), the step of sequencing the amplified nucleic acid template may include a second linearization step. The second linearization step may involve the use of oxygen guanine glycosylase (Ogg). The second linearization step may be performed after (e.g., immediately after) the step of generating a complementary form of the amplified nucleic acid template.

[0195] Preferably, the oxyguanine glycosylase is a thermophilic oxyguanine glycosylase. More preferably, the oxyguanine glycosylase is derived from a thermophilic organism, such as Methanococcus jannaschii.

[0196] In the case of performing a second sequencing read (e.g., for paired end reading), the step of sequencing the amplified nucleic acid template can include a second step of dehybridizing the complementary strand bound to (the complementary form of) the nucleic acid template with a dehybridizing agent. The dehybridizing agent can be configured to cause the complementary strand to disengage from (the complementary form of) the nucleic acid template, thereby allowing the complementary strand to be washed away. The second step of dehybridizing the complementary strand can be performed after the step of amplifying the nucleic acid template. For example, the second step of dehybridizing the complementary strand can be performed after (e.g., immediately after) the second linearization step.

[0197] Where a second sequencing read is performed (e.g., for paired end reading), the step of sequencing the amplified nucleic acid template may include a second step of hybridizing a sequencing primer to (a complementary form of) the nucleic acid template. The second step of hybridizing the sequencing primer may be performed after the step of amplifying the nucleic acid template. For example, the second step of hybridizing the sequencing primer may be performed after (e.g., immediately after) the second step of dehybridizing the complementary strands.

[0198] Where a second sequencing read is performed (e.g., for paired end reading), the step of sequencing the amplified nucleic acid template may include a second step of performing sequencing by synthesis. The second step of performing sequencing by synthesis may be performed after the step of amplifying the nucleic acid template. For example, the second step of performing sequencing by synthesis may be performed after (e.g., immediately after) the second step of hybridizing the sequencing primers.

[0199] The present disclosure will now be described by way of the following non-limiting examples.

[0200] Example

[0201] Materials and methods :

[0202] Tth PPK1 and Mru PPK2 :

[0203] Tth PPK1 and Mru PPK2 were screened to identify the conditions for soluble protein expression. Tth PPK1 and Mru PPK2 plasmids (pET28a; which added an in-frame N-terminal six-histidine purification tag) were placed in E. coli BL21 pLysS strains and spread on Luria Bertani (LB) agar plates supplemented with 50 μg / ml kanamycin antibiotics for selection. Single colonies of each corresponding transformant were picked and inoculated into LB broth supplemented with 50 μg / ml kanamycin. LB broth cultures were incubated overnight at 225 rpm at 37 ° C. Overnight cultures were used as starting cultures and added to Terrific Broth (TB) medium supplemented with 50 μg / ml kanamycin with 1% inoculum. TB cultures were incubated at 37 ° C at 225 rpm until the optical density (OD) 600 nm reached 0.6. Take out 5ml aliquots of the culture for comparative analysis of the uninduced state of the recombinant gene of interest. Induce 37°C temperature conditions with 1mM final concentration of isopropyl thiogalactoside (IPTG) and incubate for another three hours at 37°C with 225rpm. Incubate the 18°C ​​temperature condition culture in an ice water bath for 20 minutes and then induce with 1mM final concentration of isopropyl thiogalactoside (IPTG). These cultures are rotated and incubated at 18°C ​​with 225rpm for 16 hours.

[0204] Harvest culture at their corresponding time and by centrifugal precipitation.Pour out supernatant, and cells are frozen at -80 ℃, then thawed to 22 ℃.Use the lysozyme enzymatic lysis precipitation in the resuspension buffer (50mM TrispH 8.0,500mM NaCl, 25mM imidazole and 0.1Triton-X100) supplemented at 22 ℃ incubated on a rocking platform.The material of the cracking is centrifuged at 4 ℃ for 20 minutes with 18,000xg.Remove supernatant (soluble fraction), and 4 μ l is added to 20 μ l lysis buffer and 6 μ l 5X SDS loading buffer.By taking P20 pipette tip and suctioning about 1 μ l precipitation and resuspending in 24 μ l lysis buffer and 6 μ l 5X SDS loading buffer to carry out the analysis of insoluble fraction. Samples were heated to 95 °C for 5 min, cooled to 22 °C, centrifuged and loaded into SDS-PAGE gel with a 4%-12% gradient.

[0205] Mru PPK2 is used for purification. 18 ℃ overnight induction conditions are immobilized metal affinity chromatography (IMAC) for purification. After lysis and clarification, the supernatant is applied to Ni-NTA purification column. Collect flow-through (FT) and wash the column with 10 column volumes (CV) of 50mM Tris pH 8.0, 200mM NaCl and 25mM imidazole. Collect washing liquid for subsequent analysis. Elute Mru PPK2 from IMAC column with 50mM Tris pH 8.0, 200mM NaCl and 500mM imidazole. Eluted fractions, FT and washing fractions are evaluated by SDS-PAGE (same procedure as above). The fractions with eluted protein are dialyzed against 20mMTris pH 7.5, 300mM NaCl, 0.5mM DTT, 1mM EDTA and 50% glycerol and stored at -20 ℃ for activity determination.

[0206] Reference Example 1 - Effect of using pyrophosphatase to reduce PPi (Figure 5)

[0207] On-board cluster generation (OBCG) was performed using a NextSeq 2000 with a custom recipe to pull out ExAmp supplemented with 0.3U PPiase / 100μl clustering reagent or 1.2UPPiase / 100μl clustering reagent from a unique position within the sequencing box. A TruSeq Nano 450 (NA12878; source genomic DNA) supplemented with 1% PhiX v3 control at a concentration of 300pM was the inoculation library. For each test condition, two high output (HO) P3 flow cells and accompanying boxes were used. A single high output (HO) P3 flow cell was used as a control for comparison. 2X151 sequencing runs were performed for each flow cell in the flow cell. The main metrics were taken from the sequence analysis viewer (SAV). The run was analyzed using DRAGEN Germline Alignmentv3.7.5 (downsample-bam, Firebrand R&D) through the BaseSpace analysis workflow, which was automatically performed by a wrapper in the AVATAR platform.

[0208] In summary, Reference Example 1 shows that improvements in various sequencing metrics can be obtained by reducing pyrophosphate levels.

[0209] Example 2 - Amplification using polyphosphate kinase and inorganic pyrophosphate ( Figure 6 )

[0210] In vitro reactions were performed to screen for other energy supply systems for amplification reactions, e.g. Figure 6 As shown in lanes 1 to 5 in . A 990 base pair template was used (PCR amplification template, followed by purification). 280nmThe purified template was quantified and diluted to 10 nM input stock for in vitro recombinase polymerase amplification (RPA). Forward and reverse primers were added at a final concentration of 1 μM. The template in solution was amplified in a 20 μl reaction volume with template and primers using ExAmp clustering reagent. The reaction was incubated at 37°C for 30 minutes. At 30 minutes, 3 μl was removed and added to 27 μl stop buffer (NEB 6X purple loading dye [1X2.5% Ficoll; 10 mM EDTA;

[0211] 3.3 mM Tris pH 8; 0.08% EDTA; visualizing dye] and 0.8 U proteinase K) and incubated at 55° C. for 15 minutes followed by 10 minutes at 80° C. Three microliters were loaded onto a 2.2% agarose TAE gel separated by electrophoresis and visualized by ethidium bromide staining with UV illumination.

[0212] Lane 1 is a positive control, which shows that the standard amplification mixture using creatine kinase and phosphocreatine results in a band at about 1000 bp. Lane 3 shows that PPK2 and PPi can replace the creatine kinase and phosphocreatine system as an energy system. Negative control experiments show that the removal of the various components shown in lanes 2, 4, and 5 does not result in amplification.

[0213] In summary, Example 2 shows that pyrophosphate levels can be reduced using polyphosphate kinase.

[0214] Additional Notes

[0215] While various illustrative examples have been described above, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the present disclosure. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the embodiments described herein.

[0216] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein.

[0217] Sequence Listing

[0218] SEQ ID NO.1: Thermus thermophilus (Tth) polyphosphate kinase 1 (PPK1):

[0219] HLLPEASWLQFNRRVLLQTERPDFPLLERLRFLGIWNRNLDEFFAARIAKPFLKSRRGPDHLALLQEALDQAKLARARYQNLLQEAFPRLRVLDPGELDDLDWLYFRVFLAEEVAPKTDLIPWEAAQDLSHSALYFASERYLVRLPQDLPRLVEVPGREGTYVRLGALMRWRSDLLLPEEAPLYEFRVLRLLESERVRADWNELAESLEGRQEGTPTLLVVEEGFPEAWLDALRRALGLFLEEVFALKPPLNLSLVDTLVAQGPPEWRFPPFRPERPRTFLKNPLALLGKRDVLLYHPFEDYAAVERFAEAALAEEVEEVWATLYRTGEENPLAEALIAAARKGKRVHVLLEGRARFDELLNLRWYLRLVRAGVEVLPLPERKVHAKAFLILTREGGYAHLGTGNYNPTNGHHYTDFSLFTARKEVVAEVRAFFQAMAEEKTPRLGLLRTGEGIRRLLLEAVLHEAHPKGRLILKFNHLTDPELLEALVYAASRGARVDLLVRSTLTRLHPAIRAKSLVGRFLEHARAAAFRAGGEWRVYLTSADAMPRNFQNRFELLFPVLDKEAKKKVLKVLKRQVRDDRNSFLLTPEGEKRLWGGRHDAQRL

[0220] SEQ ID NO.2: Pyrobaculum aerophilum (Pae) polyphosphate kinase 2 (PPK2):

[0221] KKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLVVRVKNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE

[0222] SEQ ID NO.3: Escherichia coli polyphosphate kinase 1 (PPK1):

[0223] MGQEKLYIEKELSWLSFNERVLQEAADKSNPLIERMRFLGIYSNNLDEFYKVRFAELKRRIIISEEQGSNSHSRHLLGKIQSRVLKADQEFDGLYNELLLEMARNQIFLINERQLSVNQQNWLRHYFKQYLRQHITPILINPDTDLVQFLKDDYTYLAVEIIRGDTIRYALLEIPSDKVPRFVNLPPEAPRRRKPMILLDNILRYCLDDIFKGFFDYDALNAYSMKMTRDAEYDLVHEMEASLMELMSSSLKQRLTAEPVRFVYQRDMPNALVEVLREKLTISRYDSIVPGGRYHNFKDFINFPNVGKANLVNKPLPRLRHIWFDKAQFRNGFDAIRERDVLLYYPYHTFEHVLELLRQASFDPSVLAIKINIYRVAKDSRIIDSMIHAAHNGKKVTVVVELQARFDEEANIHWAKRLTEAGVHVIFSAPGLKIHAKLFLISRKENGEVVRYAHIGTGNFNEKTARLYTDYSLLTADARITNEVRRVFNFIENPYRPVTFDYLMVSPQNSRRLLYEMVDREIANAQQGLPSGITLKLNNLVDKGLVDRLYAASSSGVPVNLLVRGMCSLIPNLEGISDNIRAISIVDRYLEHDRVYIFENGGDKKVYLSSADWMTRNIDYRIEVATPLLDPRLKQRVLDIIDILFSDTVKARYIDKELSNRYVPRGNRRKVRAQLAIYDYIKSLEQPE

[0224] SEQ ID NO.4: P5 sequence:

[0225] AATGATACGGCGACCACCGAGATCTACAC

[0226] SEQ ID NO.5: P7 sequence:

[0227] CAAGCAGAAGACGGCATACGAGAT

[0228] SEQ ID NO.6: P5' sequence (complementary to P5):

[0229] GTGTAGATCTCGGTGGTCGCCGTATCATT

[0230] SEQ ID NO.7: P7' sequence (complementary to P7):

[0231] ATCTCGTATGCCGTCTTCTGCTTG:

[0232] SEQ ID NO:8: RB32 UvsX with His tag:

[0233] MGSSHHHHHHSSGLVPRGSHMSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKHFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADTVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDTNCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNEGGHHHHH

[0234] SEQ ID NO:9 RB32 UvsX:

[0235] MSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKHFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADTVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDTNCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNE

[0236] SEQ ID NO:10 Thermophilic UvsX HQ:

[0237] MSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKSFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADTVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDINCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNEHQHQH

[0238] SEQ ID NO:11 Thermophilic UvsX His:

[0239] MSIADLKSRLIKASTSKMTAELTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKSFKSNMSTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADIVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDINCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNEGGHHHHH

Claims

1. An amplification composition comprising an inorganic polyphosphate and a polyphosphate kinase. 2 . The amplification composition according to claim 1 , wherein the amplification composition comprises at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, and NTP.

3. An amplification composition according to claim 1 or claim 2, wherein the amplification composition comprises a recombinase.

4. The amplification composition according to any one of claims 1 to 3, wherein the amplification composition comprises a recombinase, a single-stranded nucleotide binding protein, a polymerase and NTPs.

5. The amplification composition of any one of claims 1 to 4, wherein the amplification composition comprises the inorganic polyphosphate at a concentration of about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 2 μM to about 10 μM.

6. An amplification composition according to any one of claims 1 to 5, wherein the amplification composition comprises the polyphosphate kinase at a concentration of about 0.01 μM to about 1000 μM, about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 20 μM, or about 2 μM to about 10 μM.

7. The amplification composition of any one of claims 1 to 6, wherein the inorganic polyphosphate comprises a first inorganic polyphosphate having less than 50 phosphate residues.

8. The amplification composition of any one of claims 1 to 7, wherein the inorganic polyphosphate comprises a second inorganic polyphosphate having more than 100 phosphate residues.

9. An amplification composition according to claim 8 as dependent on claim 7, wherein the ratio of the first inorganic polyphosphate to the second inorganic polyphosphate is about 90:10 to about 10:90, about 80:20 to about 20:80, about 70:30 to about 30:70, about 60:40 to about 40:60 or about 50:

50.

10. The amplification composition according to any one of claims 1 to 9, wherein the polyphosphate kinase is a thermophilic polyphosphate kinase.

11. The amplification composition of any one of claims 1 to 10, wherein the polyphosphate kinase has an optimal operating temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

12. The amplification composition according to any one of claims 1 to 11, wherein the polyphosphate kinase is selected from the group consisting of a polyphosphate kinase of the PPK1 family, a polyphosphate kinase of the PPK2 family, and a polyphosphate kinase of the PPK3 family.

13. The amplification composition according to any one of claims 1 to 12, wherein the polyphosphate kinase comprises an amino acid sequence as defined in SEQ ID NO: 1 to 3, or a functional variant or functional fragment thereof.

14. An amplification composition according to any one of claims 1 to 13, wherein the composition does not contain PEG.

15. An amplification composition according to any one of claims 1 to 14, wherein the amplification composition comprises a buffer.

16. The amplification composition of claim 15, wherein the amplification composition is buffered to a pH of about 6.0 to about 9.0, preferably about 6.5 to about 8.8, more preferably about 7.5 to about 8.7, even more preferably about 8.3 to about 8.

6.

17. The amplification composition according to any one of claims 1 to 16, wherein the composition is a clustered composition or a sequencing-by-synthesis amplification composition or a resynthesis composition.

18. A kit comprising an inorganic polyphosphate and a polyphosphate kinase.

19. The kit according to claim 18, wherein the kit further comprises a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

20. Use of the amplification composition according to any one of claims 1 to 17 or the kit according to claim 18 or claim 19 in amplifying a nucleic acid template or in sequencing a nucleic acid sequence.

21. A method of amplifying a nucleic acid template, wherein the method comprises recycling ADP to ATP using an inorganic polyphosphate and a polyphosphate kinase.

22. The method of claim 21, wherein the method comprises adding an amplification composition according to any one of claims 1 to 17.

23. A method according to claim 21 or claim 22, wherein the method comprises adding a first polyphosphate composition and a second polyphosphate composition, wherein in the first polyphosphate composition, the amount of the first inorganic polyphosphate is relatively high relative to the amount of the second inorganic polyphosphate, and Wherein in the second polyphosphate composition, the amount of the second inorganic polyphosphate is higher relative to the amount of the first inorganic polyphosphate.

24. The method of any one of claims 21 to 23, wherein the first polyphosphate composition is added before the second polyphosphate composition.

25. The method according to any one of claims 21 to 24, wherein the amplification is performed by exclusive amplification.

26. A method for sequencing a nucleic acid sequence, wherein the method comprises: Amplifying a nucleic acid template using the method according to any one of claims 21 to 25; as well as The amplified nucleic acid template is sequenced.

27. The method of claim 26, wherein the step of sequencing the amplified nucleic acid template comprises performing a first sequencing read and a second sequencing read.

28. The method according to claim 26 or claim 27, wherein the step of sequencing the amplified nucleic acid template is performed using sequencing by synthesis technology or sequencing by ligation technology.

29. The method according to any one of claims 26 to 28, wherein the method is performed isothermally.

30. The method of any one of claims 26 to 29, wherein the method is carried out at a temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

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