Method of adjusting clustering dynamics

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 / 411,973, filed on September 30, 2022, and entitled “Methods of ModulatingClustering Kinetics,” the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to novel clustering 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_08600_PCT.xml and is 16.4 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] Pyrophosphatases have been previously described for use in sequencing reactions. For example, US 5744312 A describes a sequencing composition comprising a DNA polymerase containing a phenylalanine to tyrosine mutation in combination with a pyrophosphatase. US2012004115 A1 describes sequencing results using bead-immobilized T. litoralis and Aae PPi-ase.

[0010] However, 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).

[0011] There remains a need to develop new clustering compositions and methods that can be used to improve clustering and thereby increase the throughput and accuracy of sequencing runs. The present disclosure addresses this need. Summary of the invention

[0012] In one aspect of the present disclosure, there is provided a clustered composition comprising an inorganic pyrophosphatase (also referred to herein as PPiase).

[0013] Preferably, the composition comprises inorganic pyrophosphatase 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.

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

[0015] 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.

[0016] The polymerase may be DNA polymerase I, and the recombinase may be recombinase A.

[0017] The composition may not contain PEG.

[0018] The composition may 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 composition may be a resynthesized composition.

[0020] In another aspect, a thermophilic clustering composition is provided, wherein the composition comprises a thermophilic inorganic pyrophosphatase.

[0021] In another aspect, a mesophilic clustering composition is provided, wherein the composition comprises a mesophilic inorganic pyrophosphatase.

[0022] In another aspect, a kit is provided, comprising a clustering composition, a thermophilic clustering composition, or a mesophilic clustering composition.

[0023] The kit may further comprise a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

[0024] The clustering composition, thermophilic clustering composition, mesophilic clustering composition or kit may not comprise a primer having a length of 18 to 22 base pairs.

[0025] In another aspect, a clustering composition, a thermophilic clustering composition, or a mesophilic clustering composition for amplifying and / or sequencing a nucleic acid sequence is provided.

[0026] In another aspect, a method of amplifying a target nucleic acid template is provided, the method comprising reducing or removing inorganic pyrophosphate during clustering.

[0027] In another aspect, a method of enhancing clustering kinetics of a nucleic acid amplification reaction is provided, the method comprising removing or reducing the level of an inorganic pyrophosphatase.

[0028] The method may comprise adding a clustering composition, a thermophilic clustering composition, or a mesophilic clustering composition.

[0029] Nucleic acid clustering may be performed at a temperature of about 50°C to about 75°C, preferably about 75°C.

[0030] The method may comprise adding the clustering composition only once.

[0031] In another aspect, a method for sequencing a nucleic acid sequence is provided, wherein the method comprises amplifying a nucleic acid template using the method described herein; and sequencing the amplified nucleic acid template.

[0032] The step of sequencing the amplified nucleic acid template may include performing a first sequencing read and a second sequencing read.

[0033] The step of sequencing the amplified nucleic acid template can be performed using a sequencing-by-synthesis technique or a sequencing-by-ligation technique.

[0034] The process may be carried out at a temperature of about 50°C to about 75°C, preferably about 75°C.

[0035] 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

[0036] Figure 1A A typical clustering mixture, such as exclusion amplification (ExAmp), is shown. Clustering mixtures typically cluster 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 1B The primer extension step is shown. The primer extension step in the RPA (Recombinase-Polymerase Amplification) reaction generates PPi from the DNA polymerase. Figure 1C Shown is a reaction scheme for the enzymatic hydrolysis of inorganic pyrophosphate to orthophosphate by inorganic pyrophosphatase. In the experiments described herein, Escherichia coli (E. coli) was used for exemplary purposes.

[0037] Figure 2A .) shows five independent clustered HiSeqX v2.5 flow cells sequenced on a HiSeqX instrument following first base incorporation followed by fluorescence imaging by a Typhoon scanner to study the effect of PPiase within a clustered formulation over a time course. Two pushes of a matched control (Control-2X30) and a test condition (PPiase-2X30) were recorded for thirty minutes of incubation. Two pushes of a matched control (Control-2X20) and a test condition (PPiase-2X20) were recorded for twenty minutes of incubation. Figure 2B .) Sequence Analysis Viewer (SAV) was used to extract C1 intensity from each independent lane of the flow cell of sequencing. C1 intensity was analyzed with a paired t-test and determined with a 95% confidence interval, and statistical differences (****) were observed for two pushes that were incubated for twenty minutes in the absence (black circles) and presence of PPiase (red squares). C1 intensity was measured in relative fluorescence units (RFU). The estimated graph for each lane is shown in the right figure of pairing each test condition (red circles; presence of PPiase) and control condition (black circles; absence of PPiase). Figure 2C.) Sequence Analysis Viewer (SAV) was used to extract C1 intensity from each independent lane of the flow cell of sequencing. C1 intensity was analyzed with a paired t-test and determined with a 95% confidence interval, and statistical differences (*) were observed for two pushes that were incubated for thirty minutes in the absence (black circles) and presence of PPiase (red diamonds). C1 intensity was measured with relative fluorescence units (RFU). The estimated graph for each lane is shown in the right figure of pairing each test condition (red circles; presence of PPiase) and control condition (black circles; absence of PPiase). Figure 2D .) shows a table of the average C1 values ​​(reported in RFU) of all five circulation pools aggregated. The presence of PPiase with a concentration of 1.2U / 100μl clustering reagents shows that, relative to the absence (control), the C1 intensity of two pushes for thirty minutes (2X30) incubations increases by 12.1%. The presence of PPiase with a concentration of 1.2U / 100μl clustering reagents shows that, relative to the absence of PPiase (control), the C1 intensity of two pushes for twenty minutes (2X20) incubations increases by 12.9%. Pushing twenty minutes incubation twice and pushing thirty minutes incubation twice (2X30) in the presence of PPiase with a concentration of 1.2U / 100μl clustering reagents is compared, and 1.1% difference in C1 intensity is observed. Pushing thirty minutes incubation twice (2X30) is a standard commercially available clustering formula. PPiase is included in the clustering formulation and is pushed twice for twenty minutes to reach the C1 intensity level, indicating that the kinetics of clustering in the presence of PPiase has been enhanced. Under this condition, clustering with the same intensity level as the commercial standard can be achieved in a total of 40 minutes compared to 60 minutes for a standard commercially available formulation, indicating a 33% saving in clustering time. Figure 2E .) for accurate insertion and deletion (INDEL) secondary analysis of two pushes of thirty minute incubation (control black bar; 2X30min control) versus the presence of PPiase (red bar; 2X30min 1.2U) and two pushes of twenty minute incubation (control grey bar; 2X20min control) versus the presence of PPiase (dashed red bar; 2X20min 1.2U). Accuracy is defined as the ability to correctly identify the absence of variants or the absence of false positives (F / P). A false positive is defined as a test result indicating that a person has a specific disease or condition when the person does not have the disease or condition. Figure 2F.) for two pushes of thirty minute incubation (control black bar; 2X30min control) versus the presence of PPiase (red bar; 2X30min 1.2U) and two pushes of twenty minute incubation (control grey bar; 2X20min control) versus the presence of PPiase (dashed red bar; 2X20min 1.2U) re-calling insertion and deletion (INDEL) secondary analysis. Re-calling is defined as the ability to detect a variant known to be present or the absence of a false negative (F / N). A false negative is defined as a result indicating that a person does not have a specific disease or condition when in fact the person does. Figure 2G .) for accurate single nucleotide polymorphism (SNP) secondary analysis of two pushes for thirty minute incubation (control black bar; 2X30min control) versus the presence of PPiase (red bar; 2X30min 1.2U) and two pushes for twenty minute incubation (control grey bar; 2X20min control) versus the presence of PPiase (dashed red bar; 2X20min1.2U). Accuracy is defined as the ability to correctly identify the absence of variants or the absence of false positives (F / P). A false positive is defined as a test result indicating that a person has a specific disease or condition when the person does not have the disease or condition. Figure 2H .) for re-calling single nucleotide polymorphism (SNP) secondary analysis for two pushes of thirty minute incubation (control black bar; 2X30min control) versus the presence of PPiase (red bar; 2X30min 1.2U) and two pushes of twenty minute incubation (control grey bar; 2X20min control) versus the presence of PPiase (dashed red bar; 2X20min 1.2U). Re-calling is defined as the ability to detect a variant known to be present or the absence of a false negative (F / N). A false negative is defined as a result indicating that a person does not have a specific disease or condition when in fact the person does.

[0038] Figure 3A .) Sequence Analysis Viewer (SAV) was used to extract read 1 (R1) and read 2 (R2) intensities from the NextSeq 2000 run. The black bars are the R1 or R2 intensities of the control clustered formulations with a standard commercial formula. The pink bars are the R1 or R2 intensities of the clustered formulations supplemented with 0.3U PPiase / 100μl with a standard improved formula to be taken out from a unique well with a box. The red bars are the R1 or R2 intensities of the clustered formulations supplemented with 1.2U PPiase / 100μl with a standard improved formula to be taken out from a unique well with a box. For read 1 and read 2, the presence of PPiase increased 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 increased the intensity signals of read 1 and read 2. The unit of intensity measurement is relative fluorescence unit (RFU). Figure 3B .) Mass fractions expressed as %Q30 values ​​extracted from SAV. Black bars are standard clustering formulations; pink bars are clustering formulations supplemented with 0.3U PPiase / 100μl clustering formulations; red bars are clustering formulations 1.2U PPiase / 100μl clustering formulations. Relative to the control, the %Q30 fraction increases in the presence of PPiase in a concentration-dependent manner. Figure 3C .) Instrument yield measured as G output was extracted from SAV. Black bars are standard clustering formulations; pink bars are clustering formulations supplemented with 0.3UPPiase / 100μl clustering formulation; red bars are clustering formulations 1.2UPPiase / 100μl clustering formulation. Relative to the control, the yield of the NextSeq 2000 increased in the presence of PPiase in a concentration-dependent manner. Figure 3D .) Extract the percentage of clusters passing the filter (% PF) from SAV. Black bars are standard clustering formulations; pink bars are clustering formulations supplemented with 0.3U PPiase / 100μl clustering formulations; red bars are clustering formulations 1.2U PPiase / 100μl clustering formulations. Relative to the control, the % PF of NextSeq 2000 increased in the presence of PPiase in a concentration-dependent manner. Figure 3E .) Recalling single nucleotide polymorphism (SNP) secondary analysis for NextSeq 2000 runs. Recalling is defined as the ability to detect a variant known to be present or the absence of a false negative (F / N). A false negative is defined as a result indicating that a person does not have a specific disease or condition when in fact that person has that disease or condition. Black bars are standard clustering formulations; pink bars are clustering formulations supplemented with 0.3U PPiase / 100μl clustering formulation; red bars are clustering formulations 1.2U PPiase / 100μl clustering formulation. SNP recall was unchanged relative to control under the conditions tested. Figure 3F .) Accurate single nucleotide polymorphism (SNP) secondary analysis for NextSeq 200 runs. Accuracy is defined as the ability to correctly identify an absent variant or the absence of a false positive (F / P). A false positive is defined as a test result that indicates that a person has a specific disease or condition when the person does not have the disease or condition. The black bar is the standard clustered formulation; the pink bar is the clustered formulation supplemented with 0.3U PPiase / 100μl clustered formulation; the red bar is the clustered formulation 1.2U PPiase / 100μl clustered formulation. Under the conditions tested, SNP accuracy was unchanged relative to the control. Figure 3G.) Recalling insertion and deletion (INDEL) secondary analysis for NextSeq 2000 runs. Recalling is defined as the ability to detect a variant known to be present or the absence of a false negative (F / N). A false negative is defined as a result indicating that a person does not have a specific disease or condition when in fact that person has that disease or condition. Black bars are standard clustered formulations; pink bars are clustered formulations supplemented with 0.3U PPiase / 100μl clustered formulations; red bars are clustered formulations 1.2U PPiase / 100μl clustered formulations. INDEL recall was unchanged relative to control under the conditions tested. Figure 3H .) Accurate insertion and deletion (INDEL) secondary analysis for NextSeq 200 runs. Accuracy is defined as the ability to correctly identify the absence of variants or the absence of false positives (F / P). A false positive is defined as a test result that indicates that a person has a specific disease or condition when the person does not have the disease or condition. The black bar is the standard clustered formulation; the pink bar is the clustered formulation supplemented with 0.3U PPiase / 100μl clustered formulation; the red bar is the clustered formulation 1.2U PPiase / 100μl clustered formulation. Under the conditions tested, INDEL accuracy was unchanged relative to the control.

[0039] Figure 4A .) Four independent clustered HiSeqX v2.5 flow cells were sequenced on a HiSeqX instrument following first base incorporation followed by fluorescence imaging by a Typhoon scanner to investigate the impact of PPiase within the clustered formulation in a single push 90 minute recipe configuration. HiSeqX v2.5 flow cells with lanes 1-8 are annotated as follows: 1.) 30minX2 control 2.) 1X90min with buffer blank 3.) 1X90min; 4.) 1X90min; 5.) 1X90min 6.) 1X90min with 0.3U PPiase / 100ul clustering reagent); 7.) 1X90min with 0.3U PPiase / 100ul clustering reagent); 8.) 1X90min with 0.3U PPiase / 100ul clustering reagent). Figure 4B.) Sequence Analysis Viewer (SAV) is used to extract C1 intensity from each independent lane of the flow cell of sequencing. C1 intensity is analyzed with paired t-test, and with 95% confidence interval, for 1.2U PPiase (red bar) incubated for 90 minutes per 100 μl concentration, compared to the absence of PPiase clustering reagent (gray bar) when incubated for 90 minutes, statistical difference (***) is observed. In addition, when compared with 1X90 control (gray bar), there is no significant difference between the buffer blank 1X90 containing only storage buffer and PPiase enzyme, to determine the impact of bringing storage buffer into clustering formulation (gray bar-dotted line). This also shows that under the test conditions, the PPiase enzyme drives the change of clustering solution. When compared with 2X30min standard formula control, 1.2U PPiase incubated for 90 minutes per 100 μl concentration is also not statistically significant when analyzed. This indicates that when incubated for 90 minutes in the presence of PPiase in a single push system, similar levels of intensity can be obtained in clustering reactions. Therefore, a single push of clustering reagent can achieve similar levels of intensity when supplemented with PPiase when using two pushes of reagent, which under the conditions tested would result in a reduction of reagent and affect COG.

[0040] Figure 5A .) Clustered HiSeqX v2.5 flow cells were sequenced on a HiSeqX instrument following the first base incorporation followed by fluorescence imaging by the HiSeqX scanner to investigate the impact of PPiase within the clustered formulation in a single push 60 minute formulation configuration while varying the concentration of dNTPs. The lane layout is as follows: Lane 1: Control standard ExAmp (2 pushes, 30 minutes each); Lane 2: ExAmp formulated with 0.3 mM dNTPs; Lane 3: ExAmp formulated with 0.3 mM dNTPs and 1.2 U PPiase / 100 μl ExAmp cluster mix; Lane 4: ExAmp formulated with 0.6 mM dNTPs; Lane 5: ExAmp formulated with 0.6 mM dNTPs and 1.2 U PPiase / 100 μl ExAmp cluster mix. i Lane 6: ExAmp prepared with 1.2 mM dNTPs; Lane 7: ExAmp prepared with 1.2 mM dNTPs and 1.2 U PPiase / 100 μl ExAmp clustering mixture; Lane 8: ExAmp prepared with 2.4 mM dNTPs and 1.2 U PPiase / 100 μl ExAmp clustering mixture i ase / 100 μl ExAmp clustering mix. Figure 5B.) Sequence Analysis Viewer (SAV) is used to extract C1 intensity from each independent swimming lane of the flow cell of sequencing. The black bar represents 2 pushes (2X30 Cont) of the standard control that each incubates for 30 minutes. The gray bar represents that there is no PPiase in the 60-minute time process of a single push clustering reagent under different dNTP concentrations. As dNTP concentration increases in clustering reaction, there is a downward trend in C1 concentration. However, in the presence of PPiase of 1.2U / 100μl clustered formulations, it is observed that C1 intensity signal increases with each test concentration of dNTP from 0.3mM to 1.2mM in the clustered formulations. 2.4mM dNTP concentration is not mapped because it is not paired on the flow cell. There is a relationship in the clustered formulations, wherein the dNTP concentration tends to AT coverage in the secondary metric analysis. Adding PPiase in the clustered formulations provides a way to alleviate the high concentration dNTP phenotype of low C1 intensity. DETAILED DESCRIPTION

[0041] The features described below apply to all aspects and embodiments of the present disclosure.

[0042] The present disclosure relates to amplification methods and compositions, and in particular clustering methods and compositions.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In one embodiment, the template includes a first primer binding sequence (e.g., P5, e.g., SEQ ID NO: 1), 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: 4) in the 5' to 3' direction. In another embodiment, the template includes a first primer binding site (e.g., P5', e.g., SEQ ID NO: 3, which is complementary to P5), an index sequence (e.g., i5' complementary to 15), a first sequencing binding site (e.g., SBS3' complementary to SBS3), an insert 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, e.g., SEQ ID NO: 2, 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."

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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. This is called clustering.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] Alternative sequencing methods include sequencing by ligation, for example as described in US6306597 or WO06084132, the contents of which are incorporated herein by reference.

[0063] 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.

[0064] 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 sequence determination). The first region and the second region for sequence determination are located at both 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 both ends of a single-stranded template or both ends of a double-stranded template, wherein the first region and its complement are known, and the second region and its complement are known.

[0065] 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.

[0066] 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 technology (Nucleic Acids Research 27, 24, e34 (1999)) or emulsion PCR (Science 309, 5741, 1728-1732 (2005); Nature 437, 376-380 (2005)).

[0067] A key step in nucleic acid sequencing is amplification, and in particular the generation of clusters of arrays (or clonal clusters) comprising amplified template molecules on a solid support. Amplification or clustering reactions typically use four enzymes that promote clustering, for example by isothermal systems such as recombinase-polymerase amplification or RPA (Fig. 1a). The reagents required to generate clusters as described below are referred to as clustering compositions.

[0068] As used herein, the term "cluster" may refer to a group of template polynucleotides (e.g., DNA or RNA) bound in a single hole of a circulation 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, 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 circulation 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%. This monoclonal cluster may be referred to as a clonal cluster herein.

[0069] The key step in template amplification is primer extension. Typically, 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) along with 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 ultimately cannot be sequenced. Finally, the accumulation of PPi can affect the ability to accurately call / detect variants in insertion / deletion events (INDELS) and secondary metrics.

[0070] The present disclosure provides methods for removing or reducing the amount of PPi in DNA clustering reactions. This in turn was found to improve clustering kinetics and allow amplification (and subsequent sequencing) of difficult regions of the genome.

[0071] Therefore, in one aspect of the present disclosure, an amplification cluster composition is provided, which comprises a means of reducing or removing inhibitory PPi from a system. "Reduced" means that the amount or concentration of PPi at any given time point in a system comprising the composition is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% compared to a system at the same time point that does not comprise the composition. "Removal" means that any PPi produced by a polymerase is removed / converted by the composition so that the PPI is undetectable or nearly undetectable at any given time point in the system.

[0072] Inorganic pyrophosphatase catalyzes the hydrolysis of inorganic pyrophosphate to orthophosphate. The reaction scheme is shown in Figure 1c. Inorganic pyrophosphatase removes inhibitory PPi, thereby promoting the primer extension reaction.

[0073] Thus, in one embodiment, a clustered composition comprising an inorganic pyrophosphatase is provided.

[0074] As used herein, the term "inorganic pyrophosphatase" (or inorganic diphosphatase) is an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate to orthophosphate. It will be appreciated by those skilled in the art that the inorganic pyrophosphatase may be derived from any suitable source. In one embodiment, the pyrophosphatase is derived from yeast or bacteria.

[0075] In one embodiment, the pyrophosphatase is derived from a mesophilic bacteria. Examples of mesophilic bacteria include Saccharomyces cerevisiae and Escherichia coli. In one embodiment, the inorganic pyrophosphatase comprises a sequence as shown in SEQ ID NO: 5 or a functional variant or functional fragment thereof.

[0076] MSLLNVPAGKDLPEDIYVVIEIPANADPIKYEIDKESGALFVDRFMSTAMFYPCNYGYINHTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDEAGEDAKLVAVPHSKLSKEYDHIKDVNDLPELLKAQIAHFFEHYKDLEKGKWVKVEGWENAEAAKAEIVASFERAKNK(SEQ ID NO:5)

[0077] In another embodiment, the pyrophosphatase 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, Methanopyrus, or Thermotoga. In one embodiment, the thermophile can be selected from the group consisting of Thermococcus kodacaraensis, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus species GB-D, Pyrococcus woesei, Meiothermus ruber, Thermus aquaticus, Thermus brokianus, Thermus caldophilus, Thermus filiformis, Thermus flavus, Thermococcus fumiculans, Thermococcus gorgonarius, Thermococcus litoralis, Thermotoga maritima, Thermotoga neoapollo. neopolitana and Thermus thermophilus.

[0078] In one embodiment, the thermophile is from the genus Thermus. In one embodiment, the thermophile is Thermus thermophilus and the pyrophosphatase may comprise the following sequence or a functional variant or functional fragment thereof:

[0079] MANLKSLPVGDKAPEVVHMVIEVPRGSGNKYEYDPDLGAIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGLVLSTYPLLPGVVVEVRVGLLLMEDEKGGDAKVIGVVAEDQRLDHIQDIGDVPEGVKQEIQHFFETYKALEEAKKGKWVKVTGWRDRKAAALEEVRACIARYKG(SEQ ID NO:6)

[0080] In another embodiment, the thermophile is from the genus Thermococcus. In one embodiment, the thermophile is Thermococcus littoralis and the pyrophosphatase may comprise the following sequence or a functional variant or functional fragment thereof:

[0081] MNPFHDLEPGPEVPEVVYALIEIPKGSRNKYELDKKSGLIKLDRVLYSPFYYPVDYGIIPQTWYDDDDPFDIMVIMREPTYPGVLIEARPIGLFKMIDSGDKDYKVLAVPVEDPYFNDWKDISDVPKAFLDEIAHFFQRYKELQGKEIIVEGWENAEKAKQEILRAIELYKEKFKK(SEQ ID NO:7)

[0082] In another embodiment, the thermophile is from the genus Pyrococcus. In one embodiment, the thermophile is Pyrococcus furiosus and the pyrophosphatase may comprise the following sequence or a functional variant or functional fragment thereof:

[0083] MNPFHDLEPGPDVPEVVYAIIEIPKGSRNKYELDKKTGLLKLDRVLYSPFFYPVDYGIIPRTWYEDDDPFDIMVIMREPVYPLTIIEARPIGLFKMIDSGDKDYKVLAVPVEDPYFKDWKDIDDVPKAFLDEIAHFFKRYKELQGKEIIVEGWEGEAAKREILRAIEMYKEKFGKKE(SEQ ID NO:8)

[0084] In another embodiment, the thermophile is from the genus Methanothermus. In one embodiment, the thermophile is Methanopyris kandleri and the pyrophosphatase may comprise the following sequence or a functional variant or functional fragment thereof:

[0085] MMNLWKDLEPGPNPPDVVYAVIEIPRGSRNKYEYDEERGFFKLDRVLYSPFHYPLDYGFIPRTLYDDGDPLDILVIMQDPTFPGCVIEARPIGLMKMLDDSDQDDKVLAVPTEDPRFKDVKDLDDVPKHLLDEIAHMFSEYKRLEGKETEVLGWEGADAAKEAIVHAIELYEEEHG(SEQ ID NO:9)

[0086] In one embodiment, the clustered composition comprises an inorganic pyrophosphatase 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 an inorganic pyrophosphatase 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 composition may include about 0.3U / μL, 0.4U / μL, 0.5U / μL, 0.6U / μL, 0.7U / μL, 0.8U / μL, 0.9U / μL, 1.0U / μL, 1.1U / μL, 1.2U / μL, 1.3U / μL, 1.4U / μL, 1.5U / μL, 1.6U / μL, 1.7U / μL, 1.8U / μL, 1.9U / μL or about 2.0U / μL of inorganic pyrophosphatase. In one embodiment, the composition includes a clustered composition of about 0.3U / 100μl. In another embodiment, the composition includes a clustered composition of about 1.2U / 100μl. Alternatively, the inorganic pyrophosphatase 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 composition on a dry mass basis.

[0087] As used herein, the term "inorganic pyrophosphate" (or "PPi") may refer to two phosphate residues linked by a phosphoanhydride bond.

[0088] 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).

[0089] Inorganic pyrophosphates are soluble in aqueous media.

[0090] The inventors have found that removing inorganic pyrophosphate during the clustering process, for example by adding an inorganic pyrophosphatase, has many advantages in methods for cluster generation and subsequent sequencing. First, the addition of an inorganic pyrophosphatase improves clustering kinetics. 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"). "Clustering kinetics" refers to the rate at which clonal clusters of the amplified target sequence are generated within a defined time period (e.g., a total incubation time of at least 60 minutes (30 minutes per push, and a minimum of two pushes using ExAmp)) is a typical time for clustering. Increasing cluster density is particularly important in NGS sequencing because the density of clonal clusters has a large impact on sequencing performance (e.g., data quality and total data output). Increasing cluster kinetics also leads to a reduction in clustering time (i.e., the time it takes to generate (clonal) clusters or amplify a given target sequence). This is shown, for example, in Figure 2. Here, inorganic pyrophosphatase is added to the composition for different time periods: 30 minutes and 20 minutes. As Figure 2B , Figure 2C and Figure 2D As can be seen in the Figure 3, the addition of inorganic pyrophosphatase increased the signal intensity at all time points tested compared to the lanes without inorganic pyrophosphatase. This increase in intensity was most pronounced at 20 minutes, which resulted in the same intensity as the control, which was a clustering reaction under standard conditions ( Figure 2D ). These results show that the addition of inorganic pyrophosphatase can achieve the same level of clustering (or clonal amplification of the target sequence) in about 40 minutes (compared to 60 minutes), saving about 33%. This results in a faster end-to-end turnaround time for the user. The standard time for clustering is 30 minutes. These results also show that the signal intensity after 30 minutes is much higher than the signal intensity at 30 minutes for the lane without inorganic pyrophosphatase. This result in an improved sequencing signal-to-noise ratio compared to the standard method.

[0091] Thus, this data shows that the addition of inorganic pyrophosphatase can be used to improve clustering kinetics and, in turn, reduce clustering time (and therefore turnover time) and / or increase signal intensity (and therefore sequence signal-to-noise ratio).

[0092] Improving clustering kinetics by removing or reducing PPi also leads to improved sequencing performance. FIG. 3A to FIG. 3H As shown in , the addition of 0.3U and 1.2U of inorganic pyrophosphatase increased intensity, %PF, Q30, and yield (g). "%PF" refers to the % of reads that passed 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 called incorrectly: q = -10×log10(p)". "Yield" refers to the number of bases produced in a run.

[0093] Second, 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] Third, if Figure 2E , Figure 2F , Figure 3G and Figure 3H As shown in Figure 1, these results were achieved without any adverse effects on secondary metrics. In these figures, INDEL and SNP recall (the ability to detect variants known to be present or absent with false negatives) and precision (the ability to correctly identify variants that are absent or without false positives) were measured with and without the addition of inorganic pyrophosphatase. Figure 2E , Figure 2F , Figure 3G and Figure 3H As shown in , INDEL re-calling and accuracy as well as SNP re-calling and accuracy levels were similar or identical to the control (no inorganic pyrophosphatase added).

[0095] Fourth, as mentioned above, the accumulation of inorganic pyrophosphate causes DNA polymerase (e.g., DNA polymerase) to stall. When DNA polymerase encounters structured secondary features (such as G-quadruplexes), this is problematic, causing portions of the library to not be clustered, and therefore not sequenced. The removal of inorganic pyrophosphate reduces the likelihood of polymerase stalling or prevents polymerase stalling, and therefore reduces sequence-specific errors, because the polymerase is able to cluster / structure the regions of the genome.

[0096] Fifth, in addition to improving clustering kinetics (e.g., clustering time and signal intensity), adding inorganic pyrophosphatase can also significantly reduce the amount of required clustering reagents by up to 50%. As mentioned, in clustering reactions, it may be necessary to add the 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 inventors have found that the removal of inorganic pyrophosphate significantly increases the sequence signal intensity of a single push. This is shown in Figure 4. In Figure 4, the C1 signal intensity of 2 × 30 minutes of amplification composition is compared with a single push of a composition added with inorganic pyrophosphatase. As shown in the figure, compared with the control (without the addition of inorganic pyrophosphatase), the addition of inorganic pyrophosphatase significantly increases the C1 intensity, and it is worth noting that the C1 intensity is increased compared with the 2 × 30 minutes push. Therefore, as shown in Figure 4, by increasing the incubation time to 90 minutes, a single push of a composition comprising inorganic pyrophosphatase can obtain a better intensity value than the standard double push composition for 30 minutes. Thus, by reducing PPi levels, it is possible to additionally halve the amount of composition required (ie halve the COG (cost of goods)) without compromising clustering / strength.

[0097] 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.

[0098] 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.

[0099] The composition may further comprise a recombinase. The recombinase may be a thermophilic recombinase.

[0100] 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.

[0101] In some preferred embodiments, the recombinase may be UvsX. In one embodiment, UvsX comprises or consists of SEQ ID NO: 5 or 6 or a functional fragment or functional variant thereof.

[0102] In other preferred embodiments, the recombinase may be thermophilic UvsX. In one embodiment, thermophilic UvsX comprises or consists of SEQ ID NO: 7 or 8 or a functional fragment or functional variant thereof.

[0103] The composition may further comprise a single-stranded nucleotide binding protein.

[0104] 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 gate, or functionalized variants thereof.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] The composition may also include a substrate for generating ATP.

[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 also include an ATP-generating enzyme.

[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] An ATP-generating substrate as described herein may 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 comprise creatine phosphate (CP) and creatine kinase.

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

[0116] The composition may include at least one selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. Preferably, the composition may include at least two selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. More preferably, the composition may include at least three selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. Even more preferably, the composition may include at least four selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. Even more preferably, the composition may include at least five selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation.

[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 a recombinase, a single-stranded nucleotide binding protein, a polymerase, nucleotide triphosphates (NTPs), a substrate for generating ATP, and an enzyme for generating ATP.

[0120] 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.

[0121] 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.

[0122] In another aspect, a thermophilic clustering composition is provided, wherein the composition comprises a thermophilic inorganic pyrophosphatase. The thermophilic inorganic pyrophosphatase may be derived from a thermophilic organism as described above.

[0123] In a further embodiment, the composition comprises at least one (preferably all) of a recombinase, a single-stranded DNA binding protein, a strand displacement polymerase, and an energy regenerating form.

[0124] 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.

[0125] The inorganic pyrophosphatase may have 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.

[0126] The thermophilic composition can be used for thermophilic clustering. Thermophilic clustering can utilize increasing the clustering reaction to 75°C to take advantage of the increased kinetic rate due to the Arrhenius equation. Therefore, the increased kinetics has the potential to reduce clustering time.

[0127] In another aspect, a mesophilic clustered composition is provided, wherein the composition comprises a mesophilic inorganic pyrophosphatase. The mesophilic inorganic pyrophosphatase may be derived from a mesophilic organism, such as Saccharomyces cerevisiae or Escherichia coli as described above.

[0128] 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 about 30°C to about 40°C.

[0129] Thus, in another embodiment, the inorganic pyrophosphatase may have an optimal operating 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.

[0130] 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.

[0131] 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 an inorganic pyrophosphatase is capable of catalyzing the hydrolysis of inorganic pyrophosphate to orthophosphate.

[0132] 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.

[0133] 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%, %, 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 inorganic pyrophosphatase as described above. The sequence identity of the variant can be determined using many sequence alignment programs known in the art. 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).

[0134] 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, the functional fragment may retain the catalytic activity of an inorganic pyrophosphatase as described above.

[0135] In one embodiment, the composition may not contain PEG.

[0136] In another embodiment, the composition may also or alternatively not comprise luciferase and / or apyrase and / or luciferin.

[0137] 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.

[0138] The composition can be provided in a dry form (e.g., a freeze-dried form or a lyophilized form). In this case, the composition can be rehydrated, e.g., with water or a buffer solution, before being used for clustering. In other embodiments, the composition can be provided as a solution (e.g., as an aqueous solution).

[0139] 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.

[0140] In another aspect, the disclosure relates to a kit comprising a clustered composition comprising an inorganic pyrophosphatase.

[0141] 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.

[0142] Preferably, the kit may comprise a clustering composition as described herein.

[0143] The kit may also comprise a recombinase as described herein. The recombinase may be provided separately from the (clustered) composition. For example, the recombinase may be in a different container from the (clustered) composition.

[0144] 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 (clustered) composition. For example, the single-stranded nucleotide binding protein may be in a different container from the (clustered) composition.

[0145] The kit may also comprise a polymerase as described herein. The polymerase may be provided separately from the (clustered) composition. For example, the polymerase may be in a different container than the (clustered) composition.

[0146] 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 (clustered) composition. For example, the nucleotide triphosphates may be in a container different from the (clustered) composition.

[0147] The kit may also comprise a substrate for generating ATP as described herein. The substrate for generating ATP may be provided separately from the (clustered) composition. For example, the substrate for generating ATP may be in a different container from the (clustered) composition.

[0148] The kit may also include an enzyme that generates ATP as described herein. The enzyme that generates ATP may be provided separately from the (clustered) composition. For example, the enzyme that generates ATP may be in a different container from the (clustered) composition.

[0149] The kit may include at least one selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. Preferably, the kit may include at least two selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. More preferably, the kit may include at least three selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. Even more preferably, the kit may include at least four selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. Even more preferably, the kit may include at least five selected from the group consisting of: recombinase, single-stranded nucleotide binding protein, polymerase, nucleotide triphosphate (NTP), substrate for ATP generation and enzyme for ATP generation. One or more (e.g., each of these components) can be provided separately from the (clustered) composition. For example, one or more (e.g., each of these components) can be in a different container from the (clustered) composition.

[0150] Preferably, the kit further comprises at least one selected from the group consisting of: a recombinase, an 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, an NTP, and a single-stranded nucleotide binding (SSB) protein. One or more (e.g., each of these components) can be provided separately from the (clustered) composition. For example, one or more (e.g., each of these components) can be in a different container from the (clustered) composition.

[0151] 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 (clustered) composition. For example, one or more (e.g., each of these components) may be in a different container from the (clustered) composition.

[0152] Preferably, the kit may comprise a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP), a substrate for generating ATP, and an enzyme for generating ATP. One or more (e.g., each of these components) may be provided separately from the (clustered) composition. For example, one or more (e.g., each of these components) may be in a different container from the (clustered) composition.

[0153] The kit may also include an excipient as described herein. The excipient may be provided separately from the (clustering) composition. For example, the excipient may be in a different container from the (clustering). The kit may also include one or more reagents (i.e., library preparation reagents) for preparing template nucleic acid sequences for clustering and sequencing. In one embodiment, the kit may also include an adapter sequence. The adapter sequence 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.1 (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.2 (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.3 (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.4 (P7') or a variant or fragment thereof. More preferably, the kit may comprise 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 the following items: 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 adapter sequence in the adapter sequence) can be provided separately from the (clustered) composition. For example, the adapter sequence (e.g., each adapter sequence in the adapter sequence) can be in a container different from the (clustered) composition.

[0154] 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 (clustered) composition. For example, the metal cofactor composition may be in a different container from the (clustered) composition.

[0155] 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.

[0156] In another aspect, the disclosure relates to the use of a clustering composition as described herein or a kit as described herein for clustering or sequencing nucleic acid templates.

[0157] In another aspect, a method of amplifying a nucleic acid template is provided, the method comprising reducing or removing inorganic pyrophosphate generated during clustering.

[0158] In another aspect, a method for improving clustering or increasing clustering dynamics is provided, the method comprising reducing or removing inorganic pyrophosphate produced during the clustering process. Improving clustering can mean reducing the time spent forming clusters 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.) within the genome). This improvement may be relative to clustering without reducing the level of pyrophosphate. The improvement or increase or reduction as used herein can be at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more. As shown in Figure 2, adding inorganic pyrophosphatase can achieve the same level of clustering (or clonal amplification of target sequence)-reduced by about 33% in about 40 minutes (compared to 60 minutes).

[0159] 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.

[0160] The method may comprise adding a clustering composition as defined herein to a sample containing a nucleic acid template to be clustered. The composition may be added to a sample containing a nucleic acid template to be amplified. In particular, "adding" may refer to adding the composition to a flow cell before, after or simultaneously with the sample containing the 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').

[0161] The method may include performing nucleic acid clustering at a 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, clustering may be performed at about 50°C, about 55°C, about 60°C, about 65°C, about 70°C or about 75°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. Preferably, amplification may be performed isothermally.

[0162] The method may include adding the clustering composition only once. That is, only one push of the composition is needed to generate a sufficient density of clonal clusters for subsequent sequencing. Alternatively, the composition may be added more than once, i.e., twice or more.

[0163] Amplification may be performed by exclusive amplification. Amplification may be performed by bridging amplification. In one embodiment, amplification may not be real-time PCR.

[0164] In another embodiment, the present disclosure relates to a method for sequencing a nucleic acid sequence, wherein the method comprises the steps of amplifying a nucleic acid template as described herein; and sequencing the amplified nucleic acid template.

[0165] 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.

[0166] The step of sequencing the amplified nucleic acid template may include performing a first sequencing read and a second sequencing read.

[0167] 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.

[0168] The method of sequencing a nucleic acid sequence can be performed isothermally.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] Preferably, the exonuclease is a thermophilic exonuclease. More preferably, the exonuclease is derived from a thermophilic organism, such as Pyrococcus furiosus.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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).

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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, for example, using an ATP-generating substrate and / or an ATP-generating substrate as described herein; preferably creatine kinase and / or creatine phosphate. 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.

[0181] 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.

[0182] Preferably, the oxyguanine glycosylase is a thermophilic oxyguanine glycosylase. More preferably, the oxyguanine glycosylase is derived from a thermophilic organism, such as Methanococcus jannaschii.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] The present disclosure will now be described by way of the following non-limiting examples.

[0187] Example

[0188] Embodiment 1: FIG. 2A to FIG. 2H

[0189] Cluster generation was performed using a cBOT or cBOT 2 system with a custom recipe (attached). The custom recipe was used in a time course study to examine the presence and absence of Escherichia coli (Eco) inorganic pyrophosphatase (PP i The cluster generation workflow was a single seed hybridization followed by recipe-driven amplification. TruSeq Nano 350 (NA12878; source genomic DNA) supplemented with 1% PhiX v3 control at a concentration of 300 pM was used to seed the library.

[0190] Five independent HiSeqX v2.5 flow cells were clustered. Each HiSeqX v2.5 flow cell had eight addressable lanes. The lane layout was as follows: Lane 1: Control standard ExAmp (2 pushes, 30 minutes each); Lane 2: ExAmp plus 1.2U Eco PP i ase / 100 μl ExAmp clustering mixture (2 pushes, 30 min each); Lanes 3-5: triplicate conditions, 20 min control ExAmp (2 pushes, 20 min each); Lanes 6-8: triplicate conditions, ExAmp plus 1.2 U Eco PP i ase / 100 μl ExAmp clustering mixture (2 pushes, 20 minutes each). To terminate the clustering reaction at 20 minutes, the cBOT manifold lines were physically cut and syringes were attached to the released manifold lines. 500 μl HT2 buffer was flushed into the circulation pool lanes via a syringe. Subsequently, 500 μl HT1 buffer was flushed into the circulation pool lanes via a syringe. A new manifold was replaced, and the recipe continued with linearization step 1, sequencing primer hybridization, and first base incorporation. After the first base incorporation in the Cy3 and Cy5 channels set to 450 with a PMT at a resolution of 50 μM, the circulation pool was scanned for fluorescence.

[0191] Then, 2X151 sequencing runs were performed for each of the five flow cells. The main metrics were taken from the sequence analysis viewer (SAV). The run was analyzed by DRAGEN Germline Alignment v3.7.5 (downsample-bam, Firebrand R&D) through the BaseSpace analysis workflow, which was automatically performed by the wrapper in the AVATAR platform. PrismGraphPad v 9.3.1 was used for statistical analysis for paired comparisons, and the significance confidence interval was set to <0.05.

[0192] Embodiment 2: FIG. 3A to FIG. 3H

[0193] 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.

[0194] Embodiment 3: FIG. 4A to FIG. 4B

[0195] Cluster generation was performed using a cBOT or cBOT 2 system with a custom recipe (attached). The custom recipe was used in a time course study to examine the presence and absence of Escherichia coli (Eco) inorganic pyrophosphatase (PP i The cluster generation workflow was a single seed hybridization followed by recipe-driven amplification. TruSeq Nano 350 (NA12878; source genomic DNA) supplemented with 1% PhiX v3 control at a concentration of 300 pM was used to seed the library.

[0196] Single push 90 minute time course study of clustering formulations in the presence and absence of PPiase. A.) Fluorescence scan of the flow cell after first base incorporation in the Cy3 and Cy5 channels with PMT set to 450 at a resolution of 50 μM with lanes 1-8 annotated as follows: 1.) 30 minX2 control 2.) 1X90 min with buffer blank 3.) 1X90 min; 4.) 1X90 min; 5.) 1X90 min 6.) 1X90 min with 0.3U PPiase / 100ul clustering reagent); 7.) 1X90 min with 0.3U PPiase / 100ul clustering reagent); 8.) 1X90 min with 0.3U PPiase / 100ul clustering reagent). To terminate the clustering reaction at the annotated time points, physically cut the cBOT manifold tubing and attached the syringe to the released manifold tubing. 500 μl of HT2 buffer was flushed into the flow cell lanes via syringe. Subsequently, 500 μl of HT1 buffer was flushed into the flow cell lanes via syringe. A new manifold was replaced and the recipe continued with linearization step 1, sequencing primer hybridization, and first base incorporation. A single HiSeqX v2.5 flow cell was clustered.

[0197] Then, 2X151 sequencing runs were performed for each of the four flow cells. The main metrics were taken from the sequence analysis viewer (SAV). The run was analyzed by BaseSpace analysis workflow DRAGEN Germline Alignment v3.7.5 (downsample-bam, Firebrand R&D), which was automatically performed by the wrapper in the AVATAR platform. PrismGraphPad v 9.3.1 was used for statistical analysis for paired comparisons, and the significance confidence interval was set to <0.05.

[0198] Embodiment 5: FIG. 5A to FIG. 5B

[0199] Cluster generation was performed using a cBOT or cBOT 2 system with a custom recipe (attached). The custom recipe was used in a time course study to examine the presence and absence of Escherichia coli (Eco) inorganic pyrophosphatase (PP i The cluster generation workflow was a single seed hybridization followed by recipe-driven amplification. TruSeq Nano 350 (NA12878; source genomic DNA) supplemented with 1% PhiX v3 control at a concentration of 300 pM was used to seed the library.

[0200] The HiSeqX v2.5 flow cells were clustered. Each HiSeqX v2.5 flow cell had eight addressable lanes. The lane layout was as follows: Lane 1: Control standard ExAmp (2 pushes, 30 minutes each); Lane 2: ExAmp formulated with 0.3 mM dNTPs; Lane 3: ExAmp formulated with 0.3 mM dNTPs and 1.2 U PP i Lane 4: ExAmp prepared with 0.6 mM dNTP; Lane 5: ExAmp prepared with 0.6 mM dNTP and 1.2 U PP i Lane 6: ExAmp prepared with 1.2 mM dNTP; Lane 7: ExAmp prepared with 1.2 mM dNTP and 1.2 U PP i ase / 100 μl ExAmp clustering mix; Lane 8: ExAmp prepared with 2.4 mM dNTP and 1.2 U PP i ase / 100μl ExAmp clustering mix. To terminate the clustering reaction at 60 minutes, the cBOT manifold lines were physically cut and syringes were attached to the released manifold lines. 500μl HT2 buffer was flushed into the circulation cell lanes via a syringe. Subsequently, 500μl HT1 buffer was flushed into the circulation cell lanes via a syringe. A new manifold was replaced and the recipe continued with linearization step 1, sequencing primer hybridization and first base incorporation. After the first base incorporation in the Cy3 and Cy5 channels set to 450 with a PMT at a resolution of 50μM, the circulation cell was scanned for fluorescence.

[0201] Next, a 2X151 sequencing run was performed on the flow cell.The main metrics were taken from the Sequence Analysis Viewer (SAV).

[0202] Additional Notes

[0203] 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.

[0204] 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.

[0205] Sequence Listing

[0206] SEQ ID NO:1: P5 sequence:

[0207] AATGATACGGCGACCACCGAGATCTACAC

[0208] SEQ ID NO:2 P7 sequence:

[0209] CAAGCAGAAGACGGCATACGAGAT

[0210] SEQ ID NO:3 P5' sequence (complementary to P5):

[0211] GTGTAGATCTCGGTGGTCGCCGTATCATT

[0212] SEQ ID NO:4 P7' sequence (complementary to P7):

[0213] ATCTCGTATGCCGTCTTCTGCTTG

[0214] SEQ ID NO:5:

[0215] MSLLNVPAGKDLPEDIYVVIEIPANADPIKYEIDKESGALFVDRFMSTAMFYPCNYGYINHTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDEAGEDAKLVAVPHSKLSKEYDHIKDVNDLPELLKAQIAHFFEHYKDLEKGKWVKVEGWENAEAAKAEIVASFERAKNK

[0216] SEQ ID NO:6:

[0217] MANLKSLPVGDKAPEVVHMVIEVPRGSGNKYEYDPDLGAIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGLVLSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKVIGVVAEDQRLDHIQDIGDVPEGVKQEIQHFFETYKALEAKKGKWVKVTGWRDRKAALEEVRACIARYKG

[0218] SEQ ID NO:7:

[0219] MNPFHDLEPGPEVPEVVYALIEIPKGSRNKYELDKKSGLIKLDRVLYSPFYYPVDYGIIPQTWYDDDDPFDIMVIMREPTYPGVLIEARPIGLFKMIDSGDKDYKVLAVPVEDPYFNDWKDISDVPKAFLDEIAHFFQRYKELQGKEIIVEGWENAEKAKQEILRAIELYKEKFKK

[0220] SEQ ID NO:8:

[0221] MNPFHDLEPGPDVPEVVYAIIEIPKGSRNKYELDKKTGLLKLDRVLYSPFFYPVDYGIIPRTWYEDDDPFDIMVIMREPVYPLTIIEARPIGLFKMIDSGDKDYKVLAVPVEDPYFKDWKDIDDVPKAFLDEIAHFFKRYKELQGKEIIVEGWEGAEAAKREILRAIEMYKEKFGKKE

[0222] SEQ ID NO:9:

[0223] MMNLWKDLEPGPNPPDVVYAVIEIPRGSRNKYEYDEERGFFKLDRVLYSPFHYPLDYGFIPRTLYDDGDPLDILVIMQDPTFPGCVIEARPIGLMKMLDDSDQDDKVLAVPTEDPRFKDVKDLDDVPKHLLDEIAHMFSEYKRLEGKETEVLGWEGADAAKEAIVHAIELYEEEHG

[0224] SEQ ID NO:10: RB32 UvsX with His-tag

[0225] MGSSHHHHHHSSGLVPRGSHMSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKHFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADTVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDTNCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNEGGHHHHH

[0226] SEQ ID NO:11 RB32 UvsX:

[0227] MSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKHFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADTVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDTNCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNE

[0228] SEQ ID NO:12 Thermophilic UvsX HQ:

[0229] MSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKSFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADTVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDINCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNEHQHQH

[0230] SEQ ID NO:13 Thermophilic UvsX His:

[0231] MSIADLKSRLIKASTSKMTAELTTSKFFNEKDVIRTKIPMLNIAISGAIDGGMQSGLTIFAGPSKSFKSNMSLTMVAAYLNKYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPIQSVEQLKIDMVNQLEAIERGEKVIVFIDSIGNMASKKETEDALNEKSVADMTRAKSLKSLFRIVTPYFSIKNIPCVAVNHTIETIEMFSKTVMTGGTGVMYSADIVFIIGKRQIKDGSDLQGYQFVLNVEKSRTVKEKSKFFIDVKFDGGIDPYSGLLDMALELGFVVKPKNGWYAREFLDEETGEMIREEKSWRAKDINCTTFWGPLFKHQPFRDAIKRAYQLGAIDSNEIVEAEVDELINSKVEKFKSPESKSKSAADLETDLEQLSDMEEFNEGGHHHHH

Claims

1. A clustered composition comprising an inorganic pyrophosphatase.

2. The composition of claim 1, wherein the composition comprises inorganic pyrophosphatase 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.

3. The composition according to any preceding claim, wherein the composition further comprises at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a polymerase, a nucleotide triphosphate (NTP), a substrate for generating ATP, and an enzyme for generating ATP.

4. The 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.

5. A composition according to any preceding claim, wherein the polymerase is DNA polymerase I and the recombinase is recombinase A.

6. A composition according to any preceding claim, wherein the composition does not comprise PEG.

7. A composition according to any preceding claim, wherein the 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.

8. A composition according to any preceding claim, wherein the composition is a resynthesized composition.

9. A thermophilic clustering composition, wherein the composition comprises a thermophilic inorganic pyrophosphatase.

10. A mesophilic clustering composition, wherein the composition comprises a mesophilic inorganic pyrophosphatase.

11. A kit comprising the clustering composition according to any one of claims 1 to 8, the thermophilic clustering composition according to claim 9 or the mesophilic clustering composition according to claim 10.

12. The kit according to claim 11, wherein the kit further comprises a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

13. The clustered composition according to any one of claims 1 to 8, the thermophilic clustered composition according to claim 9, the mesophilic clustered composition according to claim 10, or the kit according to claim 11, wherein the composition does not comprise primers of 18 to 22 base pairs in length.

14. Use of the clustering composition according to any one of claims 1 to 8, the thermophilic clustering composition according to claim 9 or the mesophilic clustering composition according to claim 10 for amplifying and / or sequencing nucleic acid sequences.

15. A method of amplifying a target nucleic acid template, the method comprising reducing or removing inorganic pyrophosphate during clustering.

16. A method of enhancing the clustering kinetics of a nucleic acid amplification reaction, the method comprising removing or reducing the level of inorganic pyrophosphatase.

17. The method according to claim 15 or 16, wherein the method comprises adding a clustering composition according to any one of claims 1 to 8, a thermophilic clustering composition according to claim 9 or a mesophilic clustering composition according to claim 10.

18. The method according to claim 16 or 17, wherein nucleic acid clustering is performed at a temperature of about 50°C to about 75°C, preferably about 75°C.

19. The method according to any one of claims 15 to 18, wherein the method comprises adding the clustering composition only once.

20. A method for sequencing a nucleic acid sequence, wherein the method comprises: Amplifying a nucleic acid template using the method according to claim 15; as well as The amplified nucleic acid template is sequenced.

21. The method of claim 20, wherein the step of sequencing the amplified nucleic acid template comprises performing a first sequencing read and a second sequencing read.

22. The method according to claim 20 or 21, wherein the step of sequencing the amplified nucleic acid template is performed using sequencing by synthesis technology or sequencing by ligation technology.

23. The process according to any one of claims 20 to 22, wherein the process is carried out at a temperature of about 50°C to about 75°C, preferably about 75°C.

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