Thermophilic composition for nucleic acid amplification

Abstract

The present disclosure relates to novel amplification compositions and methods, particularly for nucleic acid amplification and sequencing, preferably without involving thermophilic agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 411,965, filed on September 30, 2022, and entitled “Mesophilic Compositions for Nucleic Acid Amplification,” 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 nucleic acid amplification and sequencing, preferably without involving thermophilic agents.

[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_08500_PCT.xml and is 6.4 kilobytes in size. Background Art

[0006] The 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, measure response to various types of treatments, and whole exome sequencing, to name a few. Common techniques for detecting nucleic acid sequences in biological samples are nucleic acid amplification and sequencing.

[0007] 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.

[0008] 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.

[0009] There remains a need to develop new amplification compositions and methods that can increase the throughput and accuracy of sequencing runs. The present disclosure addresses this need. Summary of the invention

[0010] In one aspect of the present disclosure, an amplification composition (also referred to herein as a composition) comprising ATP sulfurylase is provided.

[0011] The amplification composition can include ATP sulfurylase at a concentration of about 0.01 pM to about 1000 μM, about 0.1 μM to about 500 μM, about 10 μM to about 200 μM, about 100 μM.

[0012] The amplification composition may further comprise adenosine phosphosulfate (APS). Preferably, the amplification composition comprises APS 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.

[0013] The composition may further comprise at least one selected from the group consisting of a polymerase, a recombinase, a plurality of nucleotide triphosphates (NTPs), and a single-stranded nucleotide binding (SSB) protein.

[0014] The composition may further comprise at least one selected from the group consisting of a recombinase, an NTP, and a single-stranded nucleotide binding (SSB) protein.

[0015] Preferably, the ATP sulfurylase is derived from a mesophilic organism, preferably Saccharomyces cerevisiae or E. coli. In one embodiment, the ATP sulfurylase comprises an amino acid sequence as defined in SEQ ID NO: 1 or a functional variant or fragment thereof.

[0016] Preferably, the polymerase is DNA polymerase I and the recombinase is recombinase A.

[0017] Preferably, the composition does not comprise PEG.

[0018] The amplification composition may further comprise a buffer, wherein preferably the 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.

[0019] The amplification composition can be a clustered composition or a sequencing-by-synthesis composition or a resynthesis composition.

[0020] In another aspect, there is provided a kit comprising an amplification composition as defined herein.

[0021] The kit may further comprise a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

[0022] In another aspect, an amplification composition or kit for amplifying and / or sequencing a nucleic acid sequence is provided.

[0023] In another aspect, there is provided use of an amplification composition or kit for forming monoclonal clusters of a template nucleic acid sequence.

[0024] In another aspect, a method of amplifying a target nucleic acid template is provided, the method comprising adding an amplification composition to a sample comprising the nucleic acid template.

[0025] In another aspect, a method of forming monoclonal clusters of template nucleic acid sequences is provided, the method comprising adding an amplification composition to a sample comprising the nucleic acid template.

[0026] Preferably, nucleic acid amplification is performed at a temperature between 20°C and 45°C, preferably between 30°C and 40°C.

[0027] Preferably, the method comprises adding the amplification composition only once.

[0028] In another aspect, a method for sequencing a nucleic acid sequence is provided, wherein the method comprises:

[0029] amplifying the nucleic acid template using the method described above; and sequencing the amplified nucleic acid template.

[0030] Preferably, the step of sequencing the amplified nucleic acid template comprises performing a first sequencing read and a second sequencing read.

[0031] Preferably, the step of sequencing the amplified nucleic acid template is performed using sequencing by synthesis technology or sequencing by ligation technology.

[0032] 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

[0033] Figure 1A A typical amplification mixture, such as exclusion amplification (ExAmp), is shown. An amplification mixture typically clusters library-specific DNA on a solid support (such as a flow cell) using four key enzymes: a recombinase, a DNA polymerase, a single-stranded DNA binding protein (SSB), and creatine kinase. Figure 1BThe primer extension step is shown. The primer extension step in the RPA (Recombinase-Polymerase Amplification) reaction generates PPi from the DNA polymerase.

[0034] Figure 2A Again the primer extension step in the RPA reaction is shown to generate PPi from the DNA polymerase. Figure 2B It is shown that PPi can be enzymatically converted to ATP in the presence of small molecule APS and ATP sulfurylase. Figure 2C It is shown that ATP is subsequently used by the recombinase to form filaments for homology searches.

[0035] Figure 3A and Figure 3B An overview of the strand invasion assay (SIA) is provided. A duplex is annealed, one strand (red) having a black hole quencher (BHQ) attached, and one strand (black) having a fluorescent dye (FAM) attached. An oligonucleotide (oligo; green) having a region homologous to the annealed duplex (red) is present in the reaction tube. A recombinase and (+ / -) energy regeneration system combination can be supplemented to the reaction tube. When the BHQ strand is removed by the invading unlabeled strand, causing the FAM dye to be excited, a fluorescent signal is generated.

[0036] Figure 4 HCXE is shown to support RPA reactions in the presence and absence of Sce ATP sulfurylase. M; DNA ladder markers with the sizes indicated; Lane 1 is a positive control with all components of HCXE; Lane 2 HCXE, energy system CK / CP removed, but 4.4 mM ATP present in the system; Lane 3 HCXE, energy system CK / CP removed, 4.4 mM ATP, 2 μg Sce ATP sulfurylase and 4 mM APS; Lane 4 negative control, no HCXE; PhiX template only; Lane 5 HCXE, energy system CK / CP removed, 4.4 mM ATP and 4 mM APS.

[0037] 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 formulas. The gray bars are the R1 or R2 intensities of the clustered formulations supplemented with 0.3U PPiase (pyrophosphatase) / 100μl clustered formulations taken out from unique wells with a box using a standard improved formula. The black bars are the R1 or R2 intensities of the clustered formulations supplemented with 1.2U PPiase / 100μl clustered formulations taken out from unique wells with a box using a standard improved formula. 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 signal 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 0.3U PPiase / 100μl clustered formulation; the black bar is a clustered formulation 1.2U PPiase / 100μl clustered formulation. Relative to the control, the %Q30 fraction 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 .) shows a reaction scheme for the enzymatic hydrolysis of inorganic pyrophosphate to orthophosphate by inorganic pyrophosphatase. DETAILED DESCRIPTION

[0038] The features described below apply to all aspects and embodiments of the present disclosure.

[0039] The present disclosure relates to amplification methods and compositions.

[0040] 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.

[0041] 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.

[0042] 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-500bp, but can be 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.

[0043] As used herein, an "adaptor" sequence comprises short sequence-specific oligonucleotides that are 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.

[0044] 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 non-limiting examples, double-stranded nucleic acids 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 hybridized with a complementary chain portion (e.g., a long polynucleotide chain is hybridized with a short nucleotide primer), it may still be referred to as a single-stranded nucleic acid herein.

[0045] In one embodiment, the template comprises a first primer binding sequence (e.g., P5, e.g., SEQ ID NO: 2), an index sequence (e.g., i5), a first sequencing binding site (e.g., SBS3), an insert 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', SEQ ID NO: 5) in the 5' to 3' direction. In another embodiment, the template comprises a first primer binding site (e.g., P5', SEQ ID NO: 4, which is complementary to P5), an index sequence (e.g., i5', which is complementary to I5), a first sequencing binding site (e.g., SBS3', which is complementary to SBS3), an insert sequence, a second sequencing binding site (e.g., SBS12', which is complementary to SBS12), a second index sequence (e.g., i7', which is complementary to I7), and a second primer binding sequence (e.g., P7, e.g., SEQ ID NO: 3, which is complementary to P7') in the 3' to 5' direction. Any template is referred to herein as a "template strand" or a "single-stranded template." Two template strands annealed together are referred to herein as a "double-stranded template."

[0046] 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) is 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 "clustering sequence," "clustering primer," "primer," or "cluster primer," and such terms may be used interchangeably.

[0047] In another embodiment, the P5' and P7' primer binding sequences are complementary to short primer sequences (or moss primers) 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 flow cell surface allows nucleic acid amplification. As used herein, "'" indicates a complementary strand.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 LAWN 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.

[0053] As a simple example, after attaching P5 and P7 primers, the solid support can be contacted with the template to be amplified under the conditions of allowing hybridization (or annealing, such terms can be used interchangeably) between the template and the 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°C. However, other temperatures, such as about 50°C to about 75°C, about 55°C to about 70°C or about 60°C to about 65°C, can be used during hybridization. Solid phase amplification can then be carried out. The first step of amplification is a primer extension step, in which nucleotides are added to the 3' end of the fixed primer using a template to produce a complementary chain that is fully extended. Then the template is usually washed off from the solid support. The 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. The amplification of other rounds (similar to standard PCR reactions) makes it possible to form a (monoclonal) cluster or colony of the template molecule combined with the solid support. This is called clustering.

[0054] 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" amplified products. The two chains of the amplified product will be fixed to a solid support at or near the 5' end, and this attachment is derived from the initial attachment of the amplification primer. Typically, the amplified 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] Alternative sequencing methods include sequencing by ligation, for example as described in US6306597 or WO06084132, the contents of which are incorporated herein by reference.

[0060] 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.

[0061] The present disclosure relates to a method for sequencing two regions of a target double-stranded polynucleotide template (referred to herein as the first region and the second region for sequencing). The first region and the second region for sequencing 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.

[0062] 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 comprises 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.

[0063] 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 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 technique (Nucleic Acids Research 27, 24, e34 (1999)) or emulsion PCR (Science 309, 5741, 1728-1732 (2005); Nature 437, 376-380 (2005)).

[0064] A key step in nucleic acid sequencing is amplification, and in particular the generation of clusters comprising arrays (or clonal clusters) of amplified template molecules on a solid support. The amplification or clustering reaction typically uses four enzymes that promote clustering, for example by an isothermal system such as recombinase-polymerase amplification or RPA (Fig. 1a).

[0065] The key step in template amplification is primer extension. Usually, this is carried out by polymerase, such as Bacillus subtilus (Bsu) DNA polymerase I (Pol), which produces a byproduct (as shown in Figure 1b) called inorganic pyrophosphate (PPi) by each continuous NTP (e.g., dNTP) incorporation event. The release of PPi is necessary for primer extension, but when it accumulates in the aqueous environment of in vitro reaction, it can inhibit the reaction. The accumulation of PPi can also stop DNA polymerase during chain synthesis, thereby limiting positive reaction. When polymerase encounters secondary structural features (such as G-quadruplexes) in the amplified DNA chain, this is particularly problematic. The stopping of DNA polymerase can also result in a phenotype in which the part of the library is not clustered and therefore cannot be sequenced in the end. Finally, the accumulation of PPi can affect the ability of accurately calling / detecting variants in insertion / deletion events (INDELS) and secondary metrics.

[0066] As used herein, the term "inorganic pyrophosphate" (or "PPi") may refer to two phosphate residues linked by a phosphoanhydride bond.

[0067] The inorganic pyrophosphate may be present in an acid form, a salt form, or a combination thereof. In the case where the inorganic pyrophosphate is present in a salt form, the inorganic pyrophosphate may include a cation (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).

[0068] Inorganic pyrophosphates are soluble in aqueous media.

[0069] The present disclosure provides methods for removing or reducing the amount of PPi in a clustering reaction. One method for removing PPi produced by the primer extension reaction shown in Figure 2a is to enzymatically convert PPi molecules into ATP by a transferase reaction mediated by ATP sulfurylase, as shown in Figure 2b. This reaction not only consumes the inhibitory waste product PPi, but as an additional benefit, the ATP produced can be used as an energy source for the recombinase, as shown in Figure 2c. This in turn was found to improve clustering kinetics and allow amplification (and subsequent sequencing) of difficult regions of the genome.

[0070] When the amplification composition comprises creatine kinase (CK) / creatine phosphate (CP), ATP sulfurylase / APS may also be used. Alternatively, ATP sulfurylase / APS may be used instead of creatine kinase (CK) / creatine phosphate (CP).

[0071] Therefore, in one aspect, a kind of amplification composition is provided, it comprises the means of reducing or removing inhibitory PPi from the system. By "reducing" is meant that the amount or concentration of PPi at any given time point in the system comprising the amplification composition is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% compared with the system at the same time point not comprising the amplification composition. By "removing" is meant that any PPi produced by the polymerase is removed / converted by the amplification composition, making PPi undetectable or almost undetectable at any given time point in the system.

[0072] In one embodiment, the amplification composition is a clustered composition.

[0073] 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".

[0074] The development of new clustering compositions may be more challenging as other factors need to be considered (e.g., maintaining monoclonality and high density / intensity of individual clusters).

[0075] Thus, in one aspect, an amplification (or clustering) composition comprising ATP sulfurylase is provided.

[0076] As used herein, "ATP sulfurylase" catalyzes the conversion of PPi and adenylyl sulfate (APS) to ATP (and sulfate) as follows:

[0077] APS+PPi→ATP

[0078] In one embodiment, the amplification composition comprises an ATP sulfurylase 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 amplification composition comprises an ATP sulfurylase at a concentration of about 0.01 U / μL to about 100 U / μL, about 0.1 U / μL to about 50 U / μL, about 0.2 U / μL to about 30 U / μL, about 0.3 U / μL to about 20 U / μL, about 0.5 U / μL to about 10 U / μL, or about 1.0 U / μL to about 5.0 U / μL. For example, the amplification composition can 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 ATP sulfurylase. Alternatively, the ATP sulfurylase is present in a weight % 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 % relative to the total weight % of the amplification composition on a dry mass basis.

[0079] As described herein, ATP sulfurylase is derived from a mesophilic bacterium.

[0080] As used herein, the term "mesophile" may refer to proteins that are substantially undenatured at moderate temperatures (eg, between about 20° C. and about 45° C.). These proteins may have optimal activity in the range of 30° C. to 40° C.

[0081] Thus, in another embodiment, the ATP sulfurylase may have an optimal working temperature of about 30° C. to 40° C. Preferably, the ATP sulfurylase may have an optimal working temperature of about 30° C. to about 40° C., preferably about 32° C. to about 39° C., more preferably about 34° C. to about 38° C.; for example, the ATP sulfurylase may have an optimal working temperature of about 30° C., about 35° C., about 37° C., or about 40° C.

[0082] 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.

[0083] Accordingly, the present disclosure provides a mesophilic amplification composition, wherein the amplification composition comprises a mesophilic ATP sulfurylase.

[0084] In a preferred embodiment, the ATP sulfurylase is derived from a mesophilic bacterium. Examples of mesophilic bacteria include Saccharomyces cerevisiae and Escherichia coli. In one embodiment, the ATP sulfurylase comprises a sequence as shown in SEQ ID NO: 1 or a functional variant or functional fragment thereof:

[0085] MNTALAQQIANEGGVEAWMIAQQHKSLLRFLTCGSVDDGKSTLIGRLLHDTRQIYEDQLSSLHNDSKRHGTQGEKLDLALLVDGLQAEREQGITIDVAYRYFSTEKRKFIIADT PGHEQYTRNMATGASTCELAILLIDARKGVLDQTRRHSFISTLLGIKHLVVAINKMDLVDYSEETFTRIRKDYLTFAEQLPGNLDIRFVPLSALEGDNVASQSESMPWYSGPTLL EVLETVEIQRVVDAQPMRFPVQYVNRPNLDFRGYAGTLASGRVEVGQRVKVLPSGVESNVARIVTFDGDREEAFAGEAITLVLTDEIDISRGDLLLAADEALPAVQSASVDVVW MAEQPLSPGQSYDIKIAGKKTRARVDGIRYQVDINNLTQREVENLPLNGIGLVDLTFDEPLVLDRYQQNPVTGGLIFIDRLSNVTVGAGMVHEPVSQATAAPSEFSAFELELNAL VRRHFPHWGARDLLGDK

[0086] (SEQ ID NO:1)

[0087] In another embodiment, the amplification composition further comprises 5'-adenosine phosphosulfate (APS). Thus, the amplification composition comprises ATP sulfurylase and APS.

[0088] As used herein, the term "APS" (or "5'-adenosine phosphosulfate," "adenylyl sulfate") refers to a compound comprising the following structure:

[0089]

[0090] APS can be in acid form (where the counterion can be H + ), salt forms (wherein the counter ion may be one that does not include H + cation) or a combination thereof (wherein the counter ion may be H + and excluding H +The cation may be selected from "metal cations" or "non-metal cations". 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).

[0091] In one embodiment, the amplification composition comprises an APS 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 APS is present in a weight % relative to the total weight % of the amplification 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 %.

[0092] like Figure 4 As shown, the present inventors have discovered that the generation of ATP during amplification by ATP sulfurylase provides a source of ATP for the recombinase to perform homology searching and strand invasion.

[0093] The inventors have also found that removing inorganic pyrophosphate from the amplification composition, for example by adding ATP sulfurylase, has multiple advantages in methods of nucleic acid amplification and sequencing. Specifically, the inventors have found that removing inorganic pyrophosphatase can be used to improve clustering kinetics, and in turn reduce clustering time (and therefore reduce turnaround time) and / or increase signal intensity (and therefore increase sequence signal-to-noise ratio).

[0094] 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 = -10x 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 that a reduction in PPi (such as Figure 5E As shown), the strength is increased at 0.3U and 1.2U ( Figure 5A )、%PF( Figure 5B )、Q30( Figure 5C ) and yield (g)( Figure 5D ).

[0095] 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.

[0096] 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 DNA polymerase from stagnation, and therefore reduces sequence-specific errors, because polymerase can cluster / amplify the structured regions of the genome.

[0097] Finally, in addition to improving clustering kinetics (e.g., clustering time and signal intensity), the addition of ATP sulfurylase can also significantly reduce the amount of clustering / amplification reagents required by up to 50%. As mentioned, in an amplification or clustering reaction, 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 a "push"). Multiple pushes may be necessary to achieve a desired level of sequence signal intensity. Removal of inorganic pyrophosphate can significantly increase the sequence signal intensity of a single push. Therefore, by reducing PPi levels, it is possible to additionally halve the amount of the required amplification composition (i.e., halve the COG (cost of goods)) without affecting clustering / amplification intensity.

[0098] So-called "amplification composition" refers to a composition suitable for the amplification of a target nucleic acid template. In contrast, "cluster composition" refers to a composition suitable for amplifying a (single) target sequence into a cluster as described above (that is, the composition is suitable for cluster generation, particularly suitable for producing a monoclonal cluster), not only applicable to 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.

[0099] 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 copied using bridge strand resynthesis to produce a second fixed template that is complementary to the first template. Thus, the same composition as described herein can be used for resynthesis.

[0100] The composition may also comprise a recombinant enzyme.

[0101] 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.

[0102] The composition may further comprise a single-stranded nucleotide binding protein.

[0103] 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 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 phylum, or functional variants thereof.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The composition may further comprise APS.

[0110] The composition may further comprise another ATP-generating substrate (eg, excluding APS).

[0111] As used herein, the term "ATP-generating substrate" may refer to any substrate that can react with ADP to form ATP. Examples of ATP-generating substrates include creatine phosphate (CP).

[0112] The composition may further comprise another enzyme that generates ATP (eg, excluding ATP sulfurylase).

[0113] As used herein, the term "ATP-generating enzyme" may refer to any enzyme that can catalyze the reaction of ADP forming ATP. Examples of ATP-generating enzymes include creatine kinase.

[0114] As described herein, the ATP-generating substrate can be paired with a suitable ATP-generating enzyme that catalyzes the reaction of the ATP-generating substrate with ADP to form ATP. Thus, in some preferred embodiments, the composition may include both APS and ATP sulfurylase, and / or both creatine phosphate (CP) and creatine kinase (CK).

[0115] In some embodiments, the composition may not include creatine kinase and / or creatine phosphate.

[0116] The composition may comprise ATP sulfurylase and at least one selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP), and an APS. Preferably, the composition may comprise ATP sulfurylase and at least two selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP), and an APS. More preferably, the composition may comprise ATP sulfurylase and at least three selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP), and an APS. Even more preferably, the composition may comprise ATP sulfurylase and at least four selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP), and an APS.

[0117] 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.

[0118] Preferably, the composition may comprise a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein.

[0119] Preferably, the composition may comprise ATP sulfurylase, recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP) and APS.

[0120] Preferably, the amplification composition comprises a recombinase. More preferably, the amplification composition further comprises a recombinase, a single-stranded nucleotide binding protein, a polymerase and a plurality of NTPs (eg, dNTPs).

[0121] 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.

[0122] 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.

[0123] 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 functional variant of ATP sulfurylase is capable of catalyzing the conversion of PPi and adenylyl sulfate (APS) to ATP (and sulfate).

[0124] 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.

[0125] 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%, %, 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% or at least 99% overall sequence identity, and preferably retains the catalytic activity of ATP sulfurylase as described above. The sequence identity of the variant can be determined using many sequence alignment programs known in the art. For example, the Emboss Stretcher from EMBL-EBI can be used: https: / / www.ebi.ac.uk / Tools / psa / emboss_streteher / (Default parameters were used: 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).

[0126] 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 ATP sulfurylase as described above.

[0127] In one embodiment, the amplification composition may not include PEG.

[0128] In another embodiment, the amplification composition may also or alternatively not comprise luciferase and / or apyrase and / or luciferin.

[0129] The amplification composition may also or alternatively not comprise creatine kinase (CK) and / or creatine phosphate (CP).

[0130] The amplification 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.

[0131] The amplification composition can be provided in a dried form (e.g., freeze-dried form or lyophilized 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 an aqueous solution).

[0132] 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.

[0133] In another aspect, the present disclosure relates to a kit comprising an amplification composition comprising ATP sulfurylase.

[0134] 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.

[0135] Preferably, the kit may comprise an amplification composition as described herein.

[0136] The kit may also comprise a recombinase as described herein. The recombinase may be provided separately from the (amplification) composition. For example, the recombinase may be in a different container from the (amplification) composition.

[0137] The kit may also include a single-stranded nucleotide binding protein as described herein. The single-stranded nucleotide binding protein may be provided separately from the (amplification) composition. For example, the single-stranded nucleotide binding protein may be in a different container from the (amplification) composition.

[0138] The kit may also comprise a polymerase as described herein. The polymerase may be provided separately from the (amplification) composition. For example, the polymerase may be in a different container from the (amplification) composition.

[0139] The kit may also include a variety and mixture of nucleotide triphosphates (NTPs) as described herein. The nucleotide triphosphates may be provided separately from the (amplification) composition. For example, the nucleotide triphosphates may be in a container different from the (amplification) composition.

[0140] The kit may further comprise an APS. The APS may be provided separately from the (amplification) composition. For example, the APS may be in a different container than the (amplification) composition.

[0141] The kit may also include another substrate for generating ATP as described herein (e.g., excluding APS). Another substrate for generating ATP may be provided separately from the (amplification) composition. For example, another substrate for generating ATP may be in a different container from the (amplification) composition. As described herein, another substrate for generating ATP may be creatine phosphate (CP).

[0142] The kit may also include another ATP-generating enzyme as described herein (e.g., excluding ATP sulfurylase). Another ATP-generating enzyme may be provided separately from the (amplification) composition. For example, another ATP-generating enzyme may be in a different container from the (amplification) composition. As described herein, another ATP-generating enzyme may be creatine kinase (CK).

[0143] The kit may comprise ATP sulfurylase and at least one selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP) and an APS. Preferably, the kit may comprise ATP sulfurylase and at least two selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP) and an APS. More preferably, the kit may comprise ATP sulfurylase and at least three selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP) and an APS. Even more preferably, the kit may comprise ATP sulfurylase and at least four selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP) and an APS. One or more (e.g., each of these components) may be provided separately from the (amplification) composition. For example, one or more (e.g., each of these components) may be in a container different from the (amplification) composition.

[0144] Preferably, the kit further comprises at least one selected from the group consisting of the following items: a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein. More preferably, the composition further comprises at least two selected from the group consisting of the following items: a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein. One or more (e.g., each of these components) can be provided separately from the (amplification) composition. For example, one or more (e.g., each of these components) can be in a container different from the (amplification) composition.

[0145] Preferably, the kit may comprise a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein. One or more (e.g., each of these components) may be provided separately from the (amplification) composition. For example, one or more (e.g., each of these components) may be in a different container from the (amplification) composition.

[0146] Preferably, the kit may comprise ATP sulfurylase, recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP) and APS. One or more (e.g., each of these components) may be provided separately from the (amplification) composition. For example, one or more (e.g., each of these components) may be in a different container from the (amplification) composition.

[0147] The kit may also comprise an excipient as described herein. The excipient may be provided separately from the (amplification) composition. For example, the excipient may be in a different container than the (amplification) composition.

[0148] 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. The adapter sequences may 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.2 (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.3 (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.4 (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.5 (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) may be provided separately from the (amplification) composition. For example, the adapter sequence (e.g., each of the adapter sequences) may be in a different container from the (amplification) composition.

[0149] The kit may also include a metal cofactor composition. The metal cofactor may be configured to activate one or more enzymes in the composition. For example, the metal cofactor may 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 may be provided separately from the (amplification) composition. For example, the metal cofactor composition may be in a different container from the (amplification) composition.

[0150] The kit may further comprise a solid support, preferably a flow cell. Preferably, the primers (P5 and P7) are immobilized on the flow cell as described in detail above.

[0151] In another aspect, the present disclosure relates to the use of an amplification composition as described herein or a kit as described herein for amplifying a nucleic acid template or sequencing a nucleic acid sequence.

[0152] In another aspect, a method for amplifying a nucleic acid template is provided, the method comprising reducing or removing inorganic pyrophosphate produced during nucleic acid amplification. In another aspect, a method for enhancing the amplification kinetics of a nucleic acid amplification reaction is provided, the method comprising reducing or removing inorganic pyrophosphate produced during nucleic acid amplification. The method may include adding an amplification composition to a sample containing a nucleic acid template to be amplified.

[0153] In another aspect, a method of resynthesis or improved resynthesis is provided, comprising reducing or removing inorganic pyrophosphate produced during clustering by adding a composition during the resynthesis step. Resynthesis refers to the step between a first sequencing read and a second sequencing read, wherein a template is copied using bridge strand resynthesis to produce a second fixed template complementary to the first template.

[0154] The method may include performing nucleic acid amplification 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. This is known as thermophilic clustering, and as described above, allows the user to increase clustering kinetics, reduce clustering time, and faster end-to-end time. Alternatively, amplification may be performed isothermally.

[0155] On the other hand, a method for improving clustering or enhancing clustering dynamics is provided, the method comprising removing inorganic pyrophosphate or reducing the level of inorganic pyrophosphate. Improving clustering can mean reducing the time spent forming clustering as defined above and / or increasing the density / signal intensity of clustering and / or increasing the integrity of clustering / reducing sequence-specific errors (i.e., faithful amplification of secondary structures (such as G-quadruplexes, etc.) in the genome). This improvement may be relative to clustering, without reducing the level of pyrophosphate. Improvement or increase or reduction as used herein can be at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more.

[0156] The method may comprise adding an amplification composition as defined 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').

[0157] The method may include adding the amplification composition only once. That is, only one push of the amplification composition is needed to produce a sufficient density of clonal clusters for subsequent sequencing. Alternatively, the amplification composition may be added more than once, i.e., twice or more times.

[0158] Amplification may be performed by exclusive amplification. Amplification may be performed by bridging amplification. Amplification may not be real-time PCR.

[0159] In another embodiment, the present disclosure relates to a method of sequencing a nucleic acid sequence, wherein the method comprises amplifying a nucleic acid template as described herein; and sequencing the amplified nucleic acid template.

[0160] 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.

[0161] The step of sequencing the amplified nucleic acid template may include performing a first sequencing read and a second sequencing read.

[0162] The step of sequencing the amplified nucleic acid template can be performed using sequencing by synthesis technology or sequencing by ligation technology. Preferably, the step of sequencing the amplified nucleic acid template is performed using sequencing by synthesis technology.

[0163] The method of sequencing a nucleic acid sequence can be performed isothermally.

[0164] One or more steps in the method for 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., for example, one or more steps may be performed at about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., or about 75° C. Preferably, all steps in the method for 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., for example, all steps may be performed at about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., or about 75° C.

[0165] 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.

[0166] 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.

[0167] Preferably, the exonuclease is a thermophilic exonuclease. More preferably, the exonuclease is derived from a thermophilic organism, such as Pyrococcus furiosus.

[0168] 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.

[0169] The step of sequencing the amplified nucleic acid template may include a first step of dehybridizing (or denaturing) the complementary strand bound to the nucleic acid template with a dehybridizing / denaturing agent. The dehybridizing / denaturing agent may be configured to disengage the complementary strand from the nucleic acid template, thereby allowing the complementary strand to be washed away. 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.

[0170] 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.

[0171] 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).

[0172] 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.

[0173] 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.

[0174] 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.

[0175] Where a second sequencing read is performed (e.g., for paired end reading), the step of sequencing the amplified nucleic acid may also include a 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. 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.

[0176] 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.

[0177] Preferably, the oxyguanine glycosylase is a thermophilic oxyguanine glycosylase. More preferably, the oxyguanine glycosylase is derived from a thermophilic organism, such as Methanococcus jannaschii.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] The present disclosure will now be described by way of the following non-limiting examples.

[0182] Example

[0183] Reference Example 1 - Effect of using pyrophosphatase to reduce PPi (Figure 5)

[0184] On-board cluster generation (OBCG) was performed using a NextSeq 2000 with a custom recipe to pull ExAmp supplemented with 0.3U PPiase / 100μl clustering reagent or 1.2U PPiase / 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 GermlineAlignment v3.7.5 (downsample-bam, Firebrand R&D) through the BaseSpace analysis workflow, which was automatically performed by a wrapper in the AVATAR platform.

[0185] In summary, Reference Example 1 shows that improvements in various sequencing metrics can be obtained by reducing pyrophosphate levels.

[0186] Example 2 - ATP sulfurylase can generate energy to drive the recombinase (Figure 3)

[0187] Annealed duplexes, one strand (red) having a black hole quencher (BHQ) attached and one strand (black) having a fluorescent dye (FAM) attached. An oligonucleotide (oligo; green) having a region homologous to the annealed duplex (red) is present in the reaction tube. A recombinase and (+ / -) energy regeneration system combination can be supplemented to the reaction tube. When the BHQ strand is removed by the invading unlabeled strand, causing the FAM dye to be excited, a fluorescent signal is generated.

[0188] Figure 3BATP sulfurylase is shown to generate energy to drive the recombinase.

[0189] Example 3 - ATP sulfurylase can support in vitro RPA reactions ( Figure 4 )

[0190] HCXE supports RPA reactions in the presence and absence of Sce ATP sulfurylase. M; DNA ladder markers with the sizes indicated; Lane 1 is a positive control with all components of HCXE; Lane 2 HCXE, energy system CK / CP removed, but 4.4 mM ATP present in the system; Lane 3 HCXE, energy system CK / CP removed, 4.4 mM ATP, 2 μg Sce ATP sulfurylase and 4 mM APS; Lane 4 negative control, no HCXE; PhiX template only; Lane 5 HCXE, energy system CK / CP removed, 4.4 mM ATP and 4 mM APS.

[0191] Additional Notes

[0192] 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.

[0193] 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.

Claims

1. An amplification composition comprising ATP sulfurylase.

2. The amplification composition of claim 1, wherein the amplification composition comprises ATP sulfurylase at a concentration of about 0.01 pM to about 1000 μM, about 0.1 μM to about 500 μM, about 10 μM to about 200 μM, about 100 μM.

3. An amplification composition according to any preceding claim, wherein the composition further comprises adenosine phosphosulfate (APS).

4. The amplification composition of claim 3, wherein the amplification composition comprises APS 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.

5. An amplification composition according to any preceding claim, wherein the composition further comprises at least one selected from the group consisting of a polymerase, a recombinase, a plurality of nucleotide triphosphates (NTPs), and a single-stranded nucleotide binding (SSB) protein.

6. An amplification composition according to any preceding claim, wherein the composition further comprises at least one selected from the group consisting of a recombinase, an NTP and a single-stranded nucleotide binding (SSB) protein.

7. An amplification composition according to any preceding claim, wherein the ATP sulfurylase is derived from a mesophilic organism, preferably Saccharomyces cerevisiae or Escherichia coli.

8. An amplification composition according to any preceding claim, wherein the ATP sulfurylase comprises an amino acid sequence as defined by SEQ ID NO: 1 or a functional variant or fragment thereof.

9. The amplification composition of claim 5, wherein the polymerase is DNA polymerase I and the recombinase is recombinase A.

10. An amplification composition according to any preceding claim, wherein the composition does not comprise PEG.

11. An amplification composition according to any preceding claim, wherein the amplification composition comprises a buffer, and wherein preferably the 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.

12. An amplification composition according to any preceding claim, wherein the amplification composition is a clustered composition or a sequencing-by-synthesis composition or a resynthesis composition.

13. A kit comprising an amplification composition according to any preceding claim.

14. The kit according to claim 13, wherein the kit further comprises a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

15. Use of the amplification composition according to any one of claims 1 to 12 or the kit according to claim 12 or 13 for amplifying a nucleic acid sequence and / or sequencing a nucleic acid sequence.

16. Use of the amplification composition according to any one of claims 1 to 12 or the kit according to claim 13 or 14 for forming a monoclonal cluster of a template nucleic acid sequence. 17 . A method for amplifying a target nucleic acid template, the method comprising adding the amplification composition according to any one of claims 1 to 12 to a sample containing the nucleic acid template.

18. A method of forming a monoclonal cluster of a template nucleic acid sequence, the method comprising adding the amplification composition according to any one of claims 1 to 12 to a sample comprising the nucleic acid template.

19. The method according to claim 17 or 18, wherein the nucleic acid amplification is performed at a temperature between 20°C and 45°C, preferably between 30°C and 40°C.

20. The method according to any one of claims 17 to 19, wherein the method comprises adding the amplification composition only once.

21. A method for sequencing a nucleic acid sequence, wherein the method include: Amplifying a nucleic acid template using the method according to claim 17; as well as The amplified nucleic acid template is sequenced.

22. The method of claim 21, wherein the step of sequencing the amplified nucleic acid template comprises performing a first sequencing read and a second sequencing read.

23. The method according to claim 21 or 22, wherein the step of sequencing the amplified nucleic acid template is performed using sequencing by synthesis technology or sequencing by ligation technology.

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