Resynthesis kits and methods

Abstract

The present disclosure relates to novel resynthesis kits and methods, particularly for paired sequencing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 410,162, filed on September 26, 2022, and entitled “Resynthesis Kits and Methods,” the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to novel resynthesis kits and methods, particularly for pairwise 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. This xml copy, created on September 20, 2023, is named 85491_08200_PCT.xml and is 16.1 kilobytes in size. Background Art

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

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

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

[0009] One method for sequencing a polynucleotide template involves performing paired sequencing. This involves sequencing one strand of the template (a first sequencing read, "read 1"), synthesizing a complementary form of the template (resynthesis), and then sequencing the complementary form of the template (a second sequencing read, "read 2").

[0010] While methods exist for synthesizing complementary forms of templates, there remains a need to develop new kits and methods for such resynthesis. Summary of the invention

[0011] According to one aspect of the present disclosure, a resynthesis kit comprising a thermophilic phosphatase and a polymerase is provided.

[0012] Preferably, the kit comprises thermophilic phosphatase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

[0013] Preferably, the thermophilic phosphatase is derived from a thermophilic bacterium, wherein the thermophilic bacterium belongs to the genus Pyrococcus.

[0014] Preferably, the thermophilic phosphatase comprises the amino acid sequence defined by SEQ ID NO: 1, or a functional variant or functional fragment thereof.

[0015] Preferably, the thermophilic phosphatase has a denaturation temperature of 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, 65°C or more, 70°C or more, 75°C or more, 80°C or more, 85°C or more, 90°C or more, 95°C or more, 100°C or more, 105°C or more, or 110°C or more.

[0016] Preferably, the kit comprises the polymerase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

[0017] Preferably, the polymerase is a thermophilic polymerase.

[0018] Preferably, the thermophilic polymerase has a denaturation temperature of 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, 65°C or more, 70°C or more, 75°C or more, 80°C or more, 85°C or more, 90°C or more, 95°C or more, 100°C or more, 105°C or more, or 110°C or more.

[0019] Preferably, the kit further comprises a thermophilic glycosylase, preferably a thermophilic oxoguanine glycosylase.

[0020] Preferably, the kit comprises the thermophilic glycosylase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

[0021] Preferably, the thermophilic glycosylase is derived from a thermophilic bacterium, wherein the thermophilic bacterium belongs to the genus Methanocaldococcus (Methanococcus).

[0022] Preferably, the thermophilic glycosylase comprises the amino acid sequence defined by SEQ ID NO: 2, or a functional variant or functional fragment thereof.

[0023] Preferably, the thermophilic glycosylase has a denaturation temperature of 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, 65°C or more, 70°C or more, 75°C or more, 80°C or more, 85°C or more, 90°C or more, or 95°C or more.

[0024] Preferably, the kit exonuclease, preferably a thermophilic exonuclease, preferably a thermophilic exonuclease. Preferably, the kit comprises a thermophilic exonuclease at a concentration between about 1 ug / ml and 1000 ug / ml. Preferably, the thermophilic exonuclease is derived from pyrococcus abyssi or pyrococcus furiosus. Preferably, the thermophilic exonuclease comprises an amino acid sequence defined by SEQ ID NO: 12, or a functional variant or functional fragment thereof. Preferably, the thermophilic exonuclease comprises an amino acid sequence defined by SEQ ID NO: 13, or a functional variant or functional fragment thereof.

[0025] Preferably, the thermophilic phosphatase is in a lyophilized formulation. Preferably, the lyophilized formulation comprises a thermophilic phosphatase, a salt, an exonuclease, a detergent and any one or more of magnesium chloride, magnesium acetate or magnesium sulfate. Preferably, the phosphatase comprises Thermococcus aquatica alkaline phosphatase.

[0026] Preferably, the thermophilic oxoguanine glycosylase is in a lyophilized formulation. Preferably, the lyophilized formulation comprises thermophilic oxoguanine glycosylase, salt and trehalose.

[0027] Preferably, the kit comprises at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for generating ATP, and an enzyme for generating ATP.

[0028] Preferably, the kit comprises a recombinase, a single-stranded nucleotide binding protein, nucleotide triphosphates (NTPs), a substrate for generating ATP, and an enzyme for generating ATP.

[0029] Preferably, the kit further comprises a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

[0030] Preferably, the kit further comprises instructions for using the kit in resynthesis of nucleic acid templates or paired sequencing of nucleic acid templates.

[0031] According to another aspect of the present disclosure, a resynthesis kit comprising a thermophilic glycosylase and a polymerase is provided. Preferably, the thermophilic glycosylase is as described herein. Preferably, the polymerase is as described herein.

[0032] According to yet another aspect of the present disclosure, there is provided use of the resynthesis kit according to any one of claims 1 to 17 in the resynthesis of nucleic acid templates or paired sequencing of nucleic acid sequences.

[0033] According to another aspect of the present disclosure, a method for resynthesis of a nucleotide sequence is provided, wherein the method comprises: removing a blocking phosphate group from a blocked primer using a thermophilic phosphatase to form an unblocked primer; and forming a second nucleic acid template extending from the unblocked primer using a polymerase and a first nucleic acid template.

[0034] Preferably, the thermophilic phosphatase is as defined herein.

[0035] Preferably, the polymerase is as defined herein.

[0036] Preferably, the blocked primer and / or the deblocked primer is immobilized on a solid support, preferably wherein the solid support is a flow cell.

[0037] Preferably, the step of forming a second nucleic acid template extended from the deblocked primer is performed using bridge amplification.

[0038] Preferably, the method further comprises the step of isolating the first nucleic acid template by using a thermophilic glycosylase.

[0039] Preferably, the thermophilic glycosylase is as defined herein.

[0040] Preferably, the process is carried out isothermally.

[0041] Preferably, the process is conducted at a temperature of about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C.

[0042] Preferably, the method comprises the use of a resynthesis kit as described herein.

[0043] According to another aspect of the present disclosure, a method for sequencing a nucleic acid sequence by paired sequencing is provided, wherein the method comprises: sequencing a first nucleic acid template; resynthesizing the nucleic acid sequence using the method described herein to form a second nucleic acid template; and sequencing the second nucleic acid template.

[0044] Preferably, the step of sequencing the first nucleic acid template and / or the step of sequencing the second nucleic acid template is performed using a sequencing-by-synthesis technique or a sequencing-by-ligation technique.

[0045] Preferably, the method comprises the use of a resynthesis kit as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Characterization of the Pyrococcus aquatica phosphatase is shown. The primary amino acid sequence is shown together with the predicted molecular weight without the hexa-His purification tag, which contributes approximately 2 kDa.

[0047] Figure 2 The in vitro activity of the Pyrococcus aquatica phosphatase is shown.

[0048] Figure 3 The activity range of the P. aquatica phosphatase relative to temperature is shown.

[0049] Figure 4 In vitro activity assay testing for environmental transport requirements is shown.

[0050] Figure 5 shows the activity test of the deep-sea thermococcal phosphatase in response to heat shock. (A) There was no detectable decrease in PAAP activity when incubated at 80°C for up to 70 minutes using an in vitro activity assay. (B) The nuclease activity at the corresponding time points of 20 minutes and 70 minutes was measured. For the nuclease activity assay, the samples were incubated with pUC19 DNA at a concentration of 1 mg / mL PAAP for 18 hours at 37°C. The samples were then run on a 1% agarose gel stained with Sybr gold to detect the presence of DNA. The presence of nuclease (+ control) is shown as a band or smear below the "DNA only" band. "E963" is a negative control for a nuclease-free protein preparation also incubated with pUC19. Increasing the heat shock time at 80°C reduces nuclease activity. "Abyssi NHis" is a PAAP protein.

[0051] Figure 6 Characterization and purification of the recombinant MjaOGG protein is shown. The primary amino acid sequence is shown together with the predicted molecular weight without the hexa-His purification tag which contributes approximately 2 kDa.

[0052] FIG. 7 shows the in vitro activity of the MjaOGG protein, showing activity against a single-stranded nucleic acid substrate having 8-oxo-2-deoxyguanosine modification.

[0053] Figure 8 Shown are in vitro activities assessed using the 8-oxo-2-deoxyguanosine activity assay on different concentrations of MjaOGG protein.

[0054] Fig. 9In vitro activity assay testing for environmental vessel requirements is shown. When assessed in vitro with the 8-oxo-2-deoxyguanosine activity assay, the MjaOGG enzyme retained >80% activity when heat stressed at 55°C for 10 days, and retained >70% activity when heat stressed at 37°C for 19 days. The results from these stress conditions indicate that the MjaOGG protein meets environmental vessel requirements.

[0055] Fig.10 Activity testing of the MjaOGG protein in response to heat shock is shown. When MjaOGG was incubated at 80°C for 70 minutes and then purified and stored, there was no detectable nuclease activity; see lanes 4 and 8. MjaOGG and corresponding control proteins were incubated with 1 μg of pUC19 DNA lanes 1-4 or 1 μg of λH3 DNA lanes 5-8 at 37°C for 16 hours. The samples were run on a 1% agarose gel stained with ethidium bromide to detect DNA. The presence of nuclease lanes 3 and 7 appeared as a band or smear below the DNA-only bands lanes 1 and 5. E963 is a negative control for the nuclease-free protein preparation lanes 2 and 6.

[0056] Fig.11 Thermal curves for mesophilic and thermophilic enzymes are shown; grey - current mesophilic enzymes utilized on sequencing platforms; white - thermophilic enzymes of the present disclosure.

[0057] Fig.12 Shown is a comparison between mesophilic enzymes (blue and green) and thermophilic enzymes (red) when used for resynthesis; blue - original MiniSeq recipe using mesophilic enzymes; green - adapted MiniSeq recipe for 2x250 runs, but using mesophilic enzymes; red - adapted MiniSeq recipe, but using thermophilic enzymes.

[0058] Fig.13A and Fig. 13B Shown is a comparison of E. coli exonuclease and a thermostable exonuclease during ambient shipping conditions.

[0059] Fig.14 A comparison between the activity of a thermostable exonuclease and an exonuclease derived from E. coli is shown when the enzymes are used in sequencing reactions. TEX refers to thermostable exonuclease. Novaseq Exo refers to an exonuclease derived from E. coli.

[0060] Fig.15 Shown is a comparison of the activity between the thermostable exonuclease in liquid and lyophilized form after fractionation. TEX refers to thermostable exonuclease.

[0061] Fig.16A comparison of the degradation rates of the Pyrococcus aquatica alkaline phosphatase (PAAP) in liquid and lyophilized form is shown. Lyo form refers to the lyophilized form.

[0062] Fig.17A Data showing the activity and stability of oxoguanine glycosylase (OGG) in various excipient formulations for lyophilization are presented. Fig. 17B and Fig. 17C The activity and stability of various lyophilized forms of oxoguanine glycosylase (OGG) are shown.

[0063] Fig.18 Data showing sequencing metrics using various lyophilized forms of Thermococcus aquatica alkaline phosphatase (PAAP) and oxoguanine glycosylase (OGG) are provided. DETAILED DESCRIPTION

[0064] The features described below apply to all aspects and embodiments of the present disclosure.

[0065] The present disclosure relates to resynthesis kits and methods.

[0066] The present disclosure can be used for sequencing, particularly 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.

[0067] 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.

[0068] 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.

[0069] As used herein, an "adaptor" sequence comprises a short sequence-specific oligonucleotide that is ligated to the 5' and 3' ends of each DNA (or RNA) fragment in a sequencing library as part of library preparation. An adaptor sequence may also comprise a non-peptide linker.

[0070] As will be understood by the technician, double-stranded nucleic acid will generally be formed by two complementary polynucleotide chains, which are composed of deoxyribonucleotides connected by phosphodiester bonds, but may additionally include one or more ribonucleotides and / or non-nucleotide chemical moieties and / or non-naturally occurring nucleotides and / or non-naturally occurring backbone bonds. Specifically, double-stranded nucleic acid may include non-nucleotide chemical moieties, such as joints or spacers at the 5' ends of one or two chains. As non-limiting examples, double-stranded nucleic acids may include methylated nucleotides, uracil bases, thiophosphate groups, and may also include peptide conjugates, etc. Such non-DNA or non-natural modifications may be included to give nucleic acid some desired characteristics, such as for realizing covalent attachment, non-covalent attachment or metal coordination attachment to a solid support, or serve as a spacer to position the cleavage site to the optimal distance from a solid support. Single-stranded nucleic acid is composed of one such polynucleotide chain. In the case where a polynucleotide chain is only 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.

[0071] In one embodiment, the template includes a first primer binding sequence (e.g., P5, e.g., comprising a sequence defined by SEQ ID NO: 3), an index sequence (e.g., i5), a first sequencing binding site (e.g., SBS3), an insertion sequence, a second sequencing binding site (e.g., SBS12), a second index sequence (e.g., i7), and a second primer binding sequence (e.g., P7', e.g., comprising a sequence defined by SEQ ID NO: 6) in the 5' to 3' direction. In another embodiment, the template includes a first primer binding site (e.g., P5' complementary to P5, e.g., comprising a sequence defined by SEQ ID NO: 5), an index sequence (e.g., i5' complementary to I5), a first sequencing binding site (e.g., SBS3' complementary to SBS3), an insertion sequence, a second sequencing binding site (e.g., SBS12' complementary to SBS12), a second index sequence (e.g., i7' complementary to I7), and a second primer binding sequence (e.g., P7 complementary to P7', e.g., comprising a sequence defined by SEQ ID NO: 4) in the 3' to 5' direction. Any template is referred to herein as a "template strand" or a "single-stranded template". The two template strands that anneal together are referred to herein as a "double-stranded template."

[0072] 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.

[0073] The P5' primer binding sequence and the P7' primer binding sequence are complementary to the short primer sequence (or lawn primer) present on the surface of the flow cell. The binding of P5' and P7' to their complementary sequences (P5 and P7) on, for example, the surface of the flow cell allows nucleic acid amplification. As used herein, "'" indicates a complementary strand.

[0074] 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 to 40 nucleotides, although in an embodiment, the present disclosure is not limited to sequences of this length. The exact identity of 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] Alternative sequencing methods include sequencing by ligation, for example as described in US6306597 or WO06084132, the contents of which are incorporated herein by reference.

[0086] Sequencing may include 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.

[0087] A typical starting point is a plurality of single-stranded templates attached to the same surface as a plurality of fixed primers complementary to the 3' end of the fixed template. The fixed primer can be reversibly blocked to prevent extension. The single-stranded template can be sequenced using a primer 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 fixed 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: Polony technology (Nucleic Acids Research) 27, 24, e34 (1999)) or emulsion PCR (Science 309, 5741, 1728-1732 (2005); Nature, 437, 376-380 (2005)).

[0088] In a typical method for pair sequencing, the first fixed template is covalently attached to the surface via a first fixed primer (e.g., a P5 or P7 primer). The surface also includes a second fixed primer (e.g., a P7 or P5 primer), which is blocked at the 3' end to prevent amplification and / or polymerization. Therefore, once the first fixed template has been sequenced (the first sequencing read, "read 1"), the second fixed primer may need to be unblocked to allow bridge amplification to produce a second fixed template. Typically, the phosphate group at the 3' end of the second fixed primer is used for blocking. In previous methods, a mesophilic phosphatase is typically used to unblock, which removes the phosphate blocking group from the 3' end of the second fixed primer.

[0089] Bridge amplification then allows the synthesis of a second fixed template that is complementary to the first. This process is called resynthesis.

[0090] A further step of linearization allows the first fixed template to be separated from the surface and washed off by cleaving the covalent bond between the first fixed template and the first fixed primer. In previous methods, this is usually performed using a mesophilic glycosylase. This then leaves a second fixed template that can be used for sequencing (the second sequencing read, "read 2").

[0091] The present disclosure has determined that changing the type of phosphatase used during resynthesis (ie, from a mesophilic phosphatase to a thermophilic phosphatase) provides improved sequencing metrics, particularly reduced error rates during the second sequencing read. Yet another advantage includes a reduction in overall run time.

[0092] In one embodiment, the present disclosure relates to a resynthesis kit comprising a thermophilic phosphatase and a polymerase.

[0093] As used herein, the term "thermophilic" or "thermostable" may refer to a protein that is not substantially denatured at high temperatures (e.g., 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, above 100°C, above 105°C, or above 110°C).

[0094] As used herein, the term "phosphatase" may refer to an enzyme that catalyzes the following reaction:

[0095] (X) n -(3')OP i →(X) n -(3')O-H+P i

[0096] wherein X refers to a nucleotide (e.g., a nucleotide comprising a nitrogenous base such as cytosine, guanine, adenosine, thymine or uracil), "n" refers to the total number of nucleotides in the (poly)nucleotide chain, (3')O refers to the oxygen atom at the 3' end of the (poly)nucleotide chain, and P i refers to a phosphate residue.

[0097] The resynthesis kit can include thermophilic phosphatase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

[0098] The thermophilic phosphatase may preferably be derived from a thermophilic bacterium, wherein the thermophilic bacterium belongs to the genus Thermococcus. In one embodiment, the thermophilic bacterium may be Thermococcus abyssinus.

[0099] In a preferred embodiment, the thermophilic phosphatase may comprise the following sequence or a functional variant or functional fragment thereof:

[0100] MSPSGVRNVIILIGDGMGFSQLQLTKLVYGHLNMEDFPYTGIELTDSLSGEVTDSAAA

[0101] GTAIATGVKTYNRMISTTNVTGKLVNLTTLLEIAQMLGKATGLVTTTRITHATPAVFA

[0102] SHVPDRDMEEEIARQLILHNVTVLMGGGREKFSEEVLKLAEDYGYSIVYTREDLEKVK

[0103] DGKVLGLFAEGHLPYVLDRSEEDVSLLEMTKKAIEILEKNPNGFFLMIEGGRIDHACH

[0104] ANDVASIVAETKEFDDVVGYVLDYARRRGDTLVIVLADHETGGLGIGLNYGHSVDID

[0105] SIRRIDASIEEMSKEIKSGGDIRDVIRRHTGLELTDEEVKEIEEAKNSTNKYALGNIIGEII

[0106] SKKLGVGFVSHKHTGEPVPLLAYGPGAENFVGFKHHVDTAKVIAKLMIFGDRSISFTIK

[0107] GVSKIKGDVTGDYRVDERDAYATLMLLLGDLVDTELENIADMDNNGIIDLLDVMAIL

[0108] QASS

[0109] (SEQ ID NO:1)

[0110] The thermophilic phosphatase may preferably have a denaturation temperature of 40° C. or more, 45° C. or more, 50° C. or more, 55° C. or more, 60° C. or more, 65° C. or more, 70° C. or more, 75° C. or more, 80° C. or more, 85° C. or more, 90° C. or more, 95° C. or more, 100° C. or more, 105° C. or more, or 110° C. or more. More preferably, the thermophilic phosphatase may have a denaturation temperature of about 40° C. to about 200° C., about 45° C. to about 195° C., about 45° C. to about 190° C., about 55° C. to about 185° C., about 60° C. to about 180° C., about 65° C. to about 175° C., about 70° C. to about 170° C., about 75° C. to about 165° C., about 80° C. to about 160° C., about 85° C. to about 155° C., about 90° C. to about 150° C., about 95° C. to about 145° C., or about 100° C. to about 140° C.

[0111] The resynthesis kit also includes a polymerase. Preferably, the polymerase can be a thermophilic polymerase. In some preferred embodiments, the polymerase can be a DNA polymerase. In other preferred embodiments, the polymerase can be an RNA polymerase. The polymerase can be provided separately from the thermophilic phosphatase. For example, the polymerase can be in a container different from the thermophilic phosphatase.

[0112] 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 displacement, 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.

[0113] The resynthesis kit may comprise a polymerase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

[0114] The polymerase may preferably have a denaturation temperature of 40° C. or more, 45° C. or more, 50° C. or more, 55° C. or more, 60° C. or more, 65° C. or more, 70° C. or more, 75° C. or more, 80° C. or more, 85° C. or more, 90° C. or more, 95° C. or more, 100° C. or more, 105° C. or more, or 110° C. or more. More preferably, the polymerase may have a denaturation temperature of about 40° C. to about 200° C., about 45° C. to about 195° C., about 45° C. to about 190° C., about 55° C. to about 185° C., about 60° C. to about 180° C., about 65° C. to about 175° C., about 70° C. to about 170° C., about 75° C. to about 165° C., about 80° C. to about 160° C., about 85° C. to about 155° C., about 90° C. to about 150° C., about 95° C. to about 145° C., or about 100° C. to about 140° C.

[0115] Preferably, the resynthesis kit may also include a thermophilic glycosylase. More preferably, the thermophilic glycosylase is a thermophilic oxoguanine glycosylase. The thermophilic glycosylase may be provided separately from the thermophilic phosphatase and / or the polymerase. For example, the polymerase may be in a different container from the thermophilic phosphatase and / or the polymerase.

[0116] As used herein, the term "glycosylase" may refer to an enzyme that catalyzes the removal of a nitrogenous base from one nucleotide in a (poly)nucleotide chain by cleaving the N-glycosidic bond, resulting in the formation of an apurinic / apyrimidinic site (AP site). For a DNA chain, a glycosylase may recognize any nitrogenous base (e.g., purine or pyrimidine) that is not selected from cytosine (C), guanine (G), adenine (A), and thymine (T); for an RNA chain, a glycosylase may recognize any nitrogenous base (e.g., purine or pyrimidine) that is not selected from cytosine (C), guanine (G), adenine (A), and uracil (U). Examples of typical nitrogenous bases recognized by glycosylases include oxoguanine (e.g., 8-oxoguanine) and alkyl purines.

[0117] Glycosylases may be monofunctional, such that they only have glycosylase activity (i.e., cleavage of N-glycosidic bonds) - cleavage of the phosphodiester bonds in the sugar-phosphate backbone may then occur in a non-catalytic manner by elimination. Other glycosylases may be bifunctional, such that they also have AP lyase activity by catalyzing phosphodiester bonds of (poly)nucleotide chains. Preferably, the glycosylase is bifunctional (i.e., has both glycosylase and AP lyase activity).

[0118] Inclusion of a thermophilic glycosylase in combination with a thermophilic phosphatase provides even further improved sequencing metrics, particularly reduced error rates during the second sequencing read, and further reduction in overall run time.

[0119] The resynthesis kit can include the thermophilic glycosylase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

[0120] The thermophilic glycosylase may preferably be derived from a thermophilic bacterium, wherein the thermophilic bacterium belongs to the genus Methanococcus. In one embodiment, the thermophilic bacterium may be Methanocaldococcus jannaschii.

[0121] In a preferred embodiment, the thermophilic glycosylase may comprise the following sequence or a functional variant or functional fragment thereof:

[0122] MMLIKKIEELKNSEIKDIIDKRIQEFKSFKNKSNEEWFKELCFCILTANFTAEGGIRIQKEI

[0123] GDGFLTLPREELEEKLKNLGHRFYRKRAEYIVLARRFKNIKDIVESFENEKVAREFLVR

[0124] NIKGIGYKEASHFLRNVGYDDVAIIDRHILRELYENNYIDEIPKTLSRRKYLEIENILRDI

[0125] GEEVNLKLSELDLYIWYLRTGKVLK

[0126] (SEQ ID NO:2)

[0127] The thermophilic glycosylase may preferably have a denaturation temperature of 40° C. or more, 45° C. or more, 50° C. or more, 55° C. or more, 60° C. or more, 65° C. or more, 70° C. or more, 75° C. or more, 80° C. or more, 85° C. or more, 90° C. or more, or 95° C. or more. More preferably, the thermophilic phosphatase may have a denaturation temperature of about 40° C. to about 200° C., about 45° C. to about 190° C., about 45° C. to about 180° C., about 55° C. to about 170° C., about 60° C. to about 160° C., about 65° C. to about 150° C., about 70° C. to about 140° C., about 75° C. to about 130° C., about 80° C. to about 120° C., about 85° C. to about 110° C., about 90° C. to about 105° C., or about 95° C. to about 100° C.

[0128] In yet another embodiment, the resynthesis kit comprises an exonuclease. In some embodiments, the exonuclease comprises a thermostable exonuclease. In some embodiments, the exonuclease comprises a thermostable exonuclease derived from Pyrococcus aquatica. In some embodiments, the thermostable exonuclease derived from Pyrococcus aquatica comprises the following sequence or a functional variant or functional fragment thereof:

[0129] MGSSHHHHHHSSGLVPRGSHMRIVAADTGGAVLDESLQPIGLISTVAVLVEKPYNTA

[0130] KEFLVKFSDPYNYDLSGRQAILDEINLAIELAKRTKPDVVHLDSTLGGIEVRKLDEPTI

[0131] EALSISDKGKEVWKDLSKDVQPLAKKFWEETGIEILAIGKSSVPVRIAEIYSGIFSVKW

[0132] AIEEVKKGHKDHLIIGLPRYMGVEIRDGKITGRTLDPREGNLYGEISIEVPNNIKWEVY

[0133] PNPLVRRFHIFEIWGEY

[0134] (SEQ ID NO:12)

[0135] In some embodiments, the exonuclease comprises a thermostable exonuclease derived from Pyrococcus furiosus. In some embodiments, the thermostable exonuclease derived from Pyrococcus furiosus comprises the following sequence or a functional variant or functional fragment thereof:

[0136] MGSSHHHHHHSSGLVPRGSHMRIVAADTGGAVLDETFEPIGLIATVAVLVEKPYRSA

[0137] KEVMVKYANPYDYDLTGRQAIRDEVLLAIELARKVKPDVIHLDSTLGGIELRKLDEP

[0138] TIDALGISDKGKEVWKELSKDLQPLARKFWEETNIEIVAIGKSSVPVRIAEIYAGIYSA

[0139] KWGIENVEKEGHLIIGLPRYMEVNIKDGKIIGRSLDPREGGLYGSAEVSVPEGVKWEI

[0140] YPNPVARRFMIFEIFSKR

[0141] (SEQ ID NO:13)

[0142] The concentration range of the thermostable nuclease in the resynthesis kit may preferably be between about 1ug / ml and about 100ug / ml, for example, between about 1ug / ml and 10ug / ml, between about 10ug / ml and 20ug / ml, between about 20ug / ml and 30ug / ml, between about 30ug / ml and 40ug / ml, between about 40ug / ml and 50ug / ml, between about 50ug / ml and 60ug / ml, between about 60ug / ml and 70ug / ml, between about 70ug / ml and 80ug / ml, between about 80ug / ml and 90ug / ml, or between about 90ug / ml and 100ug / ml.

[0143] The thermostable exonuclease may preferably include a melting temperature above 100°C, above 101°C, above 102°C, above 103°C, above 104°C, above 105°C, above 106°C, above 107°C, above 108°C, above 109°C, above 110°C, above 111°C, above 112°C, above 113°C, above 114°C, above 115°C, above 116°C, above 117°C, above 118°C, above 119°C or above 120°C. More preferably, the thermostable exonuclease may have a melting temperature of about 100° C. to about 120° C., about 101° C. to about 119° C., about 102° C. to about 118° C., about 103° C. to about 117° C., about 104° C. to about 116° C., about 105° C. to about 115° C., about 106° C. to about 114° C., about 107° C. to about 113° C., or about 108° C. to about 112° C. In some embodiments, the thermostable exonuclease comprises a melting temperature of about 108.3° C.

[0144] In some embodiments, the exonuclease acts to reduce background noise in the sequencing reaction by cleaving excess primer after clustering and before read 1 and / or after resynthesis during the paired-end turnaround step in preparation for read 2.

[0145] As used herein, the term "functional variant" refers to a variant polypeptide sequence or a portion of a polypeptide sequence that retains the biological function of the intact non-variant sequence. For example, a functional variant of a phosphatase is capable of catalyzing the conversion of a 3'-phosphorylated (poly)nucleotide chain to provide a dephosphorylated form of the (poly)nucleotide chain, as defined herein for the term "phosphatase"; a functional variant of a glycosylase is capable of catalyzing the removal of a nitrogenous base from one nucleotide in a (poly)nucleotide chain by cleaving the N-glycosidic bond, resulting in the formation of an apurinic / apyrimidinic site (AP site), and may further have AP lyase activity, as defined herein for the term "glycosylase".

[0146] 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.

[0147] 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%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 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 the phosphatase or glycosylase 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).

[0148] As used herein, the term "functional fragment" refers to a functionally active series of consecutive amino acids from a longer polypeptide or protein. For example, a functional fragment may retain the catalytic activity of a phosphatase or glycosylase as described herein.

[0149] The resynthesis kit may also include a recombinase. Preferably, the recombinase is a thermophilic recombinase. The recombinase may be provided separately from the thermophilic phosphatase and / or polymerase. For example, the recombinase may be in a different container from the thermophilic phosphatase and / or polymerase.

[0150] 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.

[0151] The resynthesis kit may also include a single-stranded nucleotide binding protein. Preferably, the recombinase is a thermophilic single-stranded nucleotide binding protein. The single-stranded nucleotide binding protein may be provided separately from a thermophilic phosphatase and / or a polymerase. For example, the single-stranded nucleotide binding protein may be in a container different from a thermophilic phosphatase and / or a polymerase.

[0152] 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.

[0153] The resynthesis kit may also include nucleotide triphosphates (NTPs). Preferably, nucleotide triphosphates may be deoxynucleotide triphosphates (dNTPs). More preferably, the kit includes a variety of NTPs or dNTPs, and preferably includes a mixture, such as 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. Nucleotide triphosphates may be provided separately from thermophilic phosphatase and / or polymerase. For example, nucleotide triphosphates may be in a container different from thermophilic phosphatase and / or polymerase.

[0154] 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.

[0155] 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.

[0156] The resynthesis kit may also include a substrate for generating ATP. The substrate for generating ATP may be provided separately from the thermophilic phosphatase and / or polymerase. For example, the substrate for generating ATP may be in a container different from the thermophilic phosphatase and / or polymerase.

[0157] 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).

[0158] The resynthesis kit may also include an enzyme that produces ATP. Preferably, the enzyme that produces ATP is a thermophilic enzyme that produces ATP. The enzyme that produces ATP may be provided separately from a thermophilic phosphatase and / or a polymerase. For example, the enzyme that produces ATP may be in a container different from a thermophilic phosphatase and / or a polymerase.

[0159] 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.

[0160] The ATP-generating substrate as described herein can be paired with a suitable ATP-generating enzyme that catalyzes the reaction of the ATP-generating substrate with ADP to form ATP. Thus, in some preferred embodiments, the resynthesis kit may include creatine phosphate (CP) and creatine kinase.

[0161] The resynthesis kit may include 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 producing ATP, and an enzyme for producing ATP. The resynthesis kit may include at least one selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for producing ATP, and an enzyme for producing ATP. Preferably, the kit may include at least two selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for producing ATP, and an enzyme for producing ATP. More preferably, the kit may include at least three selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for producing ATP, and an enzyme for producing ATP. Even more preferably, the kit may include at least four selected from the group consisting of: a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for producing ATP, and an enzyme for producing ATP. One or more (e.g., each of these components) can be provided separately from thermophilic phosphatase and / or polymerase. For example, one or more (e.g., each of these components) can be in a container different from thermophilic phosphatase and / or polymerase.

[0162] Preferably, the resynthesis kit further comprises at least one selected from the group consisting of: a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein. More preferably, the kit further comprises at least two selected from the group consisting of: a recombinase, NTPs and a single-stranded nucleotide binding (SSB) protein. One or more (e.g., each of these components) can be provided separately from a thermophilic phosphatase and / or a polymerase. For example, one or more (e.g., each of these components) can be in a container different from a thermophilic phosphatase and / or a polymerase.

[0163] Preferably, the resynthesis kit may include 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 a thermophilic phosphatase and / or a polymerase. For example, one or more (e.g., each of these components) may be in a container different from a thermophilic phosphatase and / or a polymerase.

[0164] Preferably, the resynthesis kit may include a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for producing ATP, and an enzyme for producing ATP. One or more (e.g., each of these components) may be provided separately from a thermophilic phosphatase and / or a polymerase. For example, one or more (e.g., each of these components) may be in a container different from a thermophilic phosphatase and / or a polymerase.

[0165] In some embodiments, the resynthesis kit 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.

[0166] The resynthesis kit may also include an excipient. An excipient may be included in a composition comprising at least one of a kit component as described herein (e.g., thermophilic phosphatase, polymerase, glycosylase, recombinase, single-stranded nucleotide binding protein, nucleotide triphosphate (NTP), a substrate for producing ATP and / or an enzyme for producing ATP). In other embodiments, an excipient may be provided separately from other kit components (e.g., thermophilic phosphatase, polymerase, glycosylase, recombinase, single-stranded nucleotide binding protein, nucleotide triphosphate (NTP), a substrate for producing ATP and / or an enzyme for producing ATP). For example, an excipient may be in a container different from other kit components (e.g., thermophilic phosphatase, polymerase, glycosylase, recombinase, single-stranded nucleotide binding protein, nucleotide triphosphate (NTP), a substrate for producing ATP and / or an enzyme for producing ATP). 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).

[0167] The resynthesis kit may also include one or more reagents for preparing template nucleic acid sequences for clustering and sequencing (i.e., library preparation reagents). In one embodiment, the kit may also include adapter sequences. The adapter sequences may be configured so that they can be attached 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.3 (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.4 (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.5 (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.6 (P7') or a variant or fragment thereof. More preferably, the kit may include at least two selected from the group consisting of the following items: a first adapter sequence, a second adapter sequence, a third adapter sequence, and a fourth adapter sequence. Even more preferably, the kit may comprise at least three selected from the group consisting of a first adapter sequence, a second adapter sequence, a third adapter sequence, and a fourth adapter sequence. Still even more preferably, the kit may comprise a first adapter sequence, a second adapter sequence, a third adapter sequence, and a fourth adapter sequence. The adapter sequence (e.g., each adapter sequence in the adapter sequence) can be provided separately from the thermophilic phosphatase and / or polymerase. For example, the adapter sequence (e.g., each adapter sequence in the adapter sequence) can be in a container different from the thermophilic phosphatase and / or polymerase.

[0168] The resynthesis kit may also include a metal cofactor composition. The metal cofactor can be configured to activate one or more enzymes in the resynthesis kit. For example, the metal cofactor can be configured to activate a recombinase and / or a polymerase. Preferably, the metal cofactor composition comprises magnesium ions (e.g., magnesium acetate, magnesium chloride). The metal cofactor composition can be provided separately from the thermophilic phosphatase and / or polymerase. For example, the metal cofactor composition can be in a different container from the thermophilic phosphatase and / or polymerase.

[0169] The resynthesis 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.

[0170] The resynthesis kit also includes instructions for using the kit in the resynthesis of nucleic acid templates or paired sequencing of nucleic acid templates. For example, the instructions can take the form of a manual, brochure or user guide.

[0171] In yet another embodiment, the present disclosure relates to a resynthesis kit comprising a thermophilic glycosylase and a polymerase.

[0172] Preferably, the thermophilic glycosylase is as described herein.

[0173] Preferably, the polymerase is as described herein.

[0174] In yet another embodiment, the present disclosure relates to the use of the resynthesis kit as described herein in the resynthesis of nucleic acid templates or paired sequencing of nucleic acid sequences.

[0175] Lyophilization of the composition

[0176] As used herein, the term "lyophilization" refers to a process in which a composition is frozen and then the product is dehydrated under low pressure. Lyophilization causes the composition to transition directly from a solid phase to a gas phase without passing through a liquid phase. As used herein, lyophilization methods include shelf freeze drying and spray freeze drying to produce lyophilized cakes and / or lyophilized microspheres.

[0177] In one aspect, any phosphatase described herein is lyophilized. In some embodiments, the lyophilized formulation comprising the phosphatase comprises any one or more of a salt, an exonuclease, a detergent, and magnesium chloride, magnesium acetate, or magnesium sulfate. In embodiments, the salt comprises sodium chloride. In embodiments, the salt comprises potassium chloride. In embodiments, the exonuclease comprises Pyrococcus aquaticus alkaline phosphatase (PAAP). In embodiments, the detergent comprises polysorbate 20 or tris(ethylene glycol) dodecyl ether.

[0178] In some embodiments, the lyophilized formulation comprising the phosphatase comprises (i) a buffer, (ii) a salt, (iii) (2-hydroxypropyl)-β-cyclodextrin (HPBCD), (iv) (tris(2-carboxyethyl(phosphine)) (TCEP), (v) polysorbate 20 or tris(ethylene glycol) dodecyl ether, (vi) trehalose, (vii) zinc chloride or zinc acetate, (viii) PAAP, (ix) magnesium chloride, magnesium acetate or magnesium sulfate. In some embodiments, the buffer comprises tris or bis tris propane buffer. In some embodiments, the buffer comprises a pH between about 7.5 and 9. In some embodiments, the salt comprises potassium chloride or sodium chloride. In some embodiments, the salt concentration is between 50mM and 200mM. In some embodiments, the concentration of HPBCD is between 0.1%% w / v and 4% w / v. In some embodiments, the concentration of TCEP is between 0.5mM and 5mM. In some embodiments, the concentration of polysorbate 20 or tris(ethylene glycol) dodecyl ether is between 0.005% w / v and 0.1% w / v. In some embodiments, the concentration of trehalose is between 4% w / v and 20% w / v. In some embodiments, the molar ratio of the concentration of zinc chloride or zinc acetate to the concentration of PAAP is between 1 and 3. In some embodiments, the concentration of PAAP is between 0.002mg / ml and 1mg / ml. In some embodiments, the concentration of chloride, acetate or sulfate is between 2mM and 20mM.

[0179] In one aspect, any glycosylase described herein is lyophilized. In some embodiments, the lyophilized formulation comprising a glycosylase comprises (i) a salt, (ii) oxoguanine glycosylase (OGG) and (iii) trehalose or trehalose and raffinose. In some embodiments, the salt comprises sodium hydroxide or potassium hydroxide.

[0180] In some embodiments, the lyophilized formulation comprising a glycosylase comprises (i) a buffer, (ii) a salt, (iii) HPBCD, (iv) TCEP, (v) polysorbate 20 or tricosyl glycol ether, (vi) trehalose, (vii) raffinose, (viii) OGG, and (ix) polyvinylpyrrolidone (PVP). In some embodiments, the buffer comprises a tris buffer having a pH between 7.5 and 9. In some embodiments, the salt comprises potassium chloride or sodium chloride. In some embodiments, the salt concentration is between 20 mM and 300 mM. In some embodiments, HPBCD is between 0.1% w / v and 3.0% w / v. In some embodiments, polysorbate 20 or tricosyl glycol ether is between 0.005% and 0.1%. In some embodiments, trehalose is between 4% w / v and 20% w / v. In some embodiments, raffinose is between 0.1% w / v and 3% w / v. In some embodiments, OGG is between 0.1 uM and 500 uM. In some embodiments, PVP is between 0.1% and 2%.

[0181] On the one hand, any thermostable exonuclease described herein is lyophilized. In some embodiments, the lyophilized formulation comprising a thermostable exonuclease comprises (i) buffer, (ii) salt, (iii) trehalose, (iv) hydroxypropyl-β-cyclodextrin, (v) magnesium acetate, magnesium sulfate or magnesium chloride, (vi) tris(ethylene glycol) dodecyl ether or polysorbate 20, and (vii) thermostable exonuclease. In some embodiments, the buffer comprises BisTris propane or Tris. In some embodiments, the pH of the buffer is between about 7.5 and 9. In some embodiments, the salt is sodium hydroxide or potassium hydroxide. In some embodiments, the concentration of the salt is between 50mM and 200mM. In some embodiments, the concentration of trehalose is between 4% w / v and 30% w / v. In some embodiments, the concentration of magnesium acetate, magnesium sulfate or magnesium chloride is between 2mM and 50mM. In some embodiments, the concentration of tricosyl glycol ether or polysorbate 20 is between 0.005% and 0.2%.

[0182] method

[0183] In yet another embodiment, the present disclosure relates to a method for resynthesis of a nucleotide sequence, wherein the method comprises: removing a blocking phosphate group from a blocked primer using a thermophilic phosphatase to form an unblocked primer; and forming a second nucleic acid template extending from the unblocked primer using a polymerase and a first nucleic acid template.

[0184] As used herein, the term "blocked phosphate group" may refer to a phosphate residue present at the end of a (poly)nucleotide chain (e.g., the 3' end of a (poly)nucleotide chain). Under the reaction conditions used during amplification and / or sequencing (e.g., a reaction using a polymerase), the blocked phosphate group may prevent chain extension from the end of the (poly)nucleotide chain.

[0185] As used herein, the term "blocked primer" may refer to an inactive form of a primer (e.g., a P5 or P7 primer as described herein) that is unable to undergo chain extension from the blocked end of its (poly)nucleotide chain (e.g., at the 3' end of the (poly)nucleotide chain) under the reaction conditions used during amplification and / or sequencing (e.g., a reaction using a polymerase). A blocked primer may comprise a blocking group for this purpose, such as a blocking phosphate group. A blocked primer may be converted to its active form by removing the blocking group to form an "unblocked primer" that is able to undergo chain extension from the (previously blocked) end of its (poly)nucleotide chain.

[0186] As used herein, the term "first nucleic acid template" may refer to a (poly)nucleotide chain that has been previously sequenced in a first sequencing read. The first end of the first nucleic acid template may be covalently attached to an adapter sequence (e.g., P5' or P7') that is complementary to a blocked primer (e.g., a P5 or P7 primer as described herein), and is therefore capable of binding to the blocked primer by base pairing. Since the first nucleic acid template has been previously sequenced, the second end of the first nucleic acid template may be covalently attached to another adapter sequence (e.g., P7' or P5', different from the adapter sequence attached to the first end of the first nucleic acid template).

[0187] When a solid support is used, the second end of the first nucleic acid template can be covalently attached to a first fixed primer (e.g., a P7 or P5 primer as described herein, wherein the first fixed primer is different from the blocked primer). In this case, the first nucleic acid template constitutes a first fixed nucleic acid template.

[0188] As used herein, the term "second nucleic acid template" may refer to a (poly)nucleotide chain to be sequenced in a second sequencing read. The second nucleic acid template may be complementary to the first nucleic acid template. One end of the second nucleic acid template may be covalently attached to a blocking primer.

[0189] When a solid support is used, the blocking primer may constitute a second fixed primer (eg, a P5 or P7 primer, different from the first fixed primer). In this case, the second nucleic acid template constitutes a second fixed nucleic acid template.

[0190] The thermophilic phosphatase may be a thermophilic phosphatase as defined herein.

[0191] The polymerase may be a polymerase as defined herein.

[0192] Preferably, the blocked primer and / or the deblocked primer may be immobilized on a solid support, preferably wherein the solid support is a flow cell.

[0193] Preferably, the step of forming a second nucleic acid template extended from the deblocked primer is performed using bridge amplification.

[0194] In a preferred embodiment, the method further comprises the step of isolating the first nucleic acid template by using a thermophilic glycosylase.

[0195] The thermophilic glycosylase may be a thermophilic glycosylase as defined herein.

[0196] Thermoglycosylase allows the covalent bond to break in the first nucleic acid template, thereby cutting the (poly) nucleotide chain. The covalent bond can be located at the first end or the second end of the first nucleic acid template, preferably the second end. This allows the first nucleic acid template to be separated (especially when a solid support is used), thereby allowing it to be dehybridized from the second nucleic acid template. The first nucleic acid template can then be washed off.

[0197] Preferably, the method is performed isothermally. In other words, each step in the method for resynthesis of a nucleic acid sequence can be performed at the same temperature.

[0198] In a preferred embodiment, the process is carried out 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, the process can be carried out at a temperature of about 50° C., about 55° C., about 60° C., about 65° C., about 70° C. or about 75° C., preferably about 65° C.

[0199] In yet another embodiment, the present disclosure relates to a method for sequencing a nucleic acid sequence by paired sequencing, wherein the method comprises: sequencing a first nucleic acid template; resynthesizing the nucleic acid sequence using a method as described herein to form a second nucleic acid template; and sequencing the second nucleic acid template.

[0200] Preferably, the step of sequencing the first nucleic acid template and / or the step of sequencing the second nucleic acid template can be performed using a sequencing-by-synthesis technique or a sequencing-by-ligation technique.

[0201] Preferably, the method comprises the use of a resynthesis kit as described herein.

[0202] The present disclosure will now be described by way of the following non-limiting examples.

[0203] Example

[0204] Materials and methods :

[0205] Preparation of Pyrococcus aquatica alkaline phosphatase (PAAP) :

[0206] The PAAP gene was cloned into a pET15b vector containing an ampicillin resistance gene (50 μg / ml). The plasmid was transformed into BL21 cells supplemented with 50 μg / ml kanamycin and grown to an optical density of 0.6. Cells were induced with 0.5 mM IPTG and induced at 18 ° C for 18 hours. Cells were harvested and lysed with 3000 U / ml of ReadyLyse and 100 U / ml of Omincleave. The clarified lysate was heat treated (Ht Tx) at 80 ° C for 70 minutes. After cooling on ice for 60 minutes, the lysate was centrifuged and applied to a HisTrap column, and gradient elution fractions were collected and combined. The eluted samples combined by HisTrap were applied to an anion exchange column, gradient elution fractions were combined, and dialyzed using 200 mM NaCl, 50 mM Tris pH 7.5 and 50% glycerol buffer.

[0207] Properties of PAAP protein :

[0208] The PAAP protein is active against single-stranded nucleic acid substrates with 3' phosphate-modified oligomers. A 50 μl reaction mixture containing 10 μM substrate with 0.006 mg / mL (about 0.1 μM) PAAP was incubated at 60°C for 5 minutes. The reaction was terminated by adding EDTA at a final concentration of 20 mM. The samples were separated using reverse phase chromatography on a Clarity 1.7 μm oligo-MS100A LC column with a HAA gradient. The control sample in the absence of PAAP protein produced a single peak with a retention time of 5.7 minutes. After the addition of PAAP protein, the product peak of 3' phosphate cleavage appeared at 5.3 minutes. The results of this in vitro activity test are shown in Figure 2 middle.

[0209] A concentration of 0.006 mg / mL (about 0.1 μM) was used in the above in vitro activity assay to produce about 50% substrate and about 50% dephosphorylated oligomer (50% activity). Using this concentration, the enzyme was incubated using a temperature range of 35°C to 95°C using a standard 5 minute reaction. The sample was then immediately quenched with EDTA and analyzed on an HPLC clarification column. The results of the activity versus temperature test are shown in Figure 3 middle.

[0210] The PAAP protein was also found to be suitable for environmental transport conditions ( Figure 4 ). Furthermore, the PAAP protein was found to be stable to heat shock, allowing the use of a nuclease inactivation procedure (Figure 5).

[0211] Preparation of Methanococcus jannaschii Oxoguanine Glycosylase (MjaOGG) :

[0212] MjaOGG is cloned into pET28c vector, which contains kanamycin resistance gene (50 μg / ml). Plasmid is transformed into BL21 cells supplemented with 50 μg / ml kanamycin, and growing to an optical density of 0.8. Cells are induced with 0.1mM IPTG, and induced at 18 ℃ for 24 hours. Harvested cells, and cracked with the ReadyLyse of 3000U / ml and the Omincleave of 100U / ml. The clarified lysate is heat treated (Ht Tx) 70 minutes at 80 ℃. After cooling on ice for 60 minutes, lysate is applied to HisTrap posts, collected and merged gradient elution fractions. The eluted samples merged by HisTrap are applied to heparin columns, merged gradient elution fractions, and dialyzed using 300mM NaCl, 50mM Tris pH 7.5, 0.25mM DTT and 50% glycerol buffer. The samples were run on 4% to 12% SDS PAGE gels with PageRuler Plus protein marker. The heparin pooled fractions were nearly homogeneous ( Figure 6 ).

[0213] Properties of the MjaOGG protein :

[0214] A 100 μl reaction mixture containing 11 μM substrate with 0.2 μM MjaOGG was incubated at 60°C for 30 seconds. The reaction was terminated by the addition of proteinase K at a final concentration of 0.4 U / μl and incubated for 5 minutes. The samples were separated by reverse phase chromatography on a Clarity 1.7 μmoligo-MS100A LC column with an HAA gradient. The control sample in the absence of MjaOGG protein produced a single peak with a retention time of 10.72 minutes. After the addition of 0.2 μM MjaOGG, two product peaks were detected, the 3′ product at 9.6 minutes and the 5′ product at 9.89 minutes. The remaining substrate peak was detected at a retention time of 10.74 minutes. The results of the in vitro activity are shown in Figure 7, while the cleavage results of the three tested concentrations are shown in Figure 8 middle.

[0215] The MjaOGG protein was also found to be suitable for environmental transport conditions ( Fig. 9 ). In addition, the MjaOGG protein was found to be stable to heat shock, allowing the use of a nuclease inactivation procedure ( Fig.10 ).

[0216] Comparative testing of mesophilic versus thermophilic enzymes :

[0217] Three different experiments were performed to determine the effect of using thermophilic enzymes (such as the PAAP protein and the MjaOGG protein) on the error rate during read 2.

[0218] As a comparative example, an experiment was run on the Illumina MiniSeq platform using the normal mesophilic enzyme recipe, while another experiment was run using the modified recipe for 2x250 runs, but still using the mesophilic enzyme. These results are shown in Fig.12 Middle (blue - normal mesophilic enzyme formulation (V.3.0); green - modified formulation and mesophilic enzyme (V.3.3) used for 2x250 runs).

[0219] The experiment was run using a modified recipe for 2x250 runs, but using PAAP protein (0.103 mg / mL; approximately 2 μM) and MjaOGG protein (0.2 μM) (red). Fig.12 As shown in , the error rate of read 1 using the thermophilic enzyme is relatively similar to the corresponding mesophilic case. However, the error rate of read 2 using the thermophilic enzyme is reduced compared to the corresponding mesophilic case.

[0220] Therefore, switching currently used mesophilic enzymes to thermophilic enzymes (such as PAAP protein and MjaOGG protein) resulted in improved primary metrics, such as reduced error rate for read 2. In addition, the total run time was reduced from 42 minutes and 14 seconds to 19 minutes and 44 seconds.

[0221] Thermostable exonucleases for storage and sequencing reactions :

[0222] Thermostable exonucleases from Pyrococcus abyssinus and Pyrococcus furiosus can be used to cleave excess flow cell primers after clustering and before read 1 and / or after resynthesis during the paired-end turnaround step in preparation for read 2 to reduce background noise / signal.

[0223] Storage of exonucleases. Exonucleases derived from E. coli are not stable enough for ambient shipping conditions. Fig.13A As shown in the figure above, exonucleases are highly active when fresh. Fig.13A As shown in the middle and lower panels of , the exonuclease activity derived from E. coli was significantly reduced after storage at 37°C for 10 days and after shaking at 600 rpm at room temperature for 10 days. Fig. 13BAs shown in, the thermostable exonucleases derived from Pyrococcus abyssinus (SEQ ID NO: 12) and Pyrococcus furiosus (SEQ ID NO: 13) retained their activity after heating at 37°C for 10 to 16 days with or without shaking. In addition, the thermostable exonucleases Pyrococcus abyssinus and Pyrococcus furiosus retained their activity through six freeze / thaw cycles. The thermostable exonucleases were each tested at two different concentrations: 0.003375 mg / ml and 0.00675 mg / ml.

[0224] Sequencing using exonucleases. The activity of the thermostable exonucleases in sequencing reactions was tested and compared to the activity of exonucleases derived from E. coli in sequencing reactions. The formulations including the thermostable exonucleases contained the following ingredients:

[0225] Tris, pH 8.9

[0226] 0.1% Polysorbate 20

[0227] 0.1 mM EDTA

[0228] ●0.05 mg / ml Thermococcus aquatica thermostable exonuclease (SEQ ID NO: 12)

[0229] 45 mM magnesium chloride

[0230] 50 mM potassium chloride

[0231] 500 mM trehalose

[0232] Fig.14 It was shown that the use of a thermostable exonuclease derived from Thermococcus aquatica was as effective as the use of an exonuclease from E. coli in sequencing reactions. TEX refers to thermostable exonuclease. Novaseq Exo refers to an exonuclease derived from E. coli.

[0233] Lyophilization of Thermostable Exonucleases. Thermostable exonucleases activity was compared between liquid and lyophilized forms at different temperatures. Thermostable exonucleases were lyophilized in the following formulations:

[0234] 80 mM Bis Tris Propane pH 9

[0235] 100 mM potassium chloride

[0236] 392mM trehalose

[0237] 4 mM tris(2-carboxyethyl)phosphine

[0238] ●0.02% triethylene glycol dodecyl ether

[0239] 2% (2-hydroxypropyl)-β-cyclodextrin

[0240] 0.44 mg / ml of Thermococcus aquatica thermostable exonuclease (SEQ ID NO: 12).

[0241] Prior to testing, the lyophilisates were resuspended with 10 mM magnesium acetate. The samples were fractionated. After fractionation in liquid and lyophilized form, the thermostable exonuclease activity was compared at different temperatures. Fig.15 Thermostable exonucleases were shown to have significantly longer shelf lives in lyophilized form, which enables storage in ambient conditions.

[0242] Degradation rate of liquid Pyrococcus alkaline phosphatase (PAAP) and lyophilized PAAP

[0243] PAAP was lyophilized in the following formulations:

[0244] 80 mM BisTris Propane pH 9

[0245] 100 mM potassium chloride

[0246] 393mM trehalose

[0247] 4 mM tris(2-carboxyethyl)phosphine

[0248] ●0.02% triethylene glycol dodecyl ether

[0249] 2% (2-hydroxypropyl)-β-cyclodextrin

[0250] 8uM zinc acetate

[0251] 4uM PAAP (SEQ ID NO: 1)

[0252] Fig.16 It was shown that there was a lower degradation rate of PAAP when the PAAP was in lyophilized form relative to when the PAAP was in liquid form.

[0253] Screening of Methanococcus jannaschii oxoguanine glycosylase (MjaOGG). MjaOGG (OGG) (SEQ ID NO: 2) was screened for activity in the presence of bulking lyophilization excipients before and after heat stress. (2-Hydroxypropyl)-β-cyclodextrin (HPBCD), polyvinylpyrrolidone (PVP), raffinose and trehalose were selected as candidates for freeze drying. Each lyophilized formulation contained the following essential components:

[0254] 50 mM HEPES pH 7.5

[0255] 300 mM sodium chloride

[0256] 250 mM trehalose

[0257] 0.05% Polysorbate 20

[0258] 7uM OGG (SEQ ID NO: 2)

[0259] Five different formulations were tested. One formulation contained only the essential components shown in the above formulation. Four additional formulations were tested: (i) each of the essential components in the above formulation and (2-hydroxypropyl)-β-cyclodextrin (HPBCD); (ii) each of the essential components in the above formulation and Maltodex; (iii) each of the essential components in the above formulation and polyvinylpyrrolidone (PVP); and (iv) each of the essential components in the above formulation and raffinose. Fig.17A The activities of preparations (i), (iii) and (iv) were shown to result in high activity of the OGG enzyme.

[0260] The activity and stability of OGG were analyzed in microsphere form and pellet form. The lyophilized formulation tested contained the following components:

[0261] 50mM Tris pH 7.5

[0262] 300 mM sodium chloride

[0263] 10.5% trehalose

[0264] 3% (2-hydroxypropyl)-β-cyclodextrin

[0265] 3% Raffinose

[0266] 0.05% Polysorbate 20

[0267] Fig. 17B and Fig. 17C It was shown that there was no difference in OGG activity between the pellet format and the microsphere format.

[0268] Lyophilized and liquid forms of PAAP (SEQ ID NO: 1) and OGG (SEQ ID NO: 2) were tested to see how the forms performed on primary and secondary sequencing metrics. The following formulations were tested;

[0269] OLM2 (liquid preparation of OGG)

[0270] 50mM Tris pH 7.5

[0271] 300 mM sodium chloride

[0272] 0.05% Polysorbate 20

[0273] 0.5 nM OGG

[0274] KLM2 (lyophilized preparation of OGG)

[0275] Tris pH 7.5

[0276] 14.75% trehalose

[0277] 4% (2-hydroxypropyl)-β-cyclodextrin

[0278] 70 mM sodium chloride

[0279] ●0.05% tris(ethylene glycol) dodecyl ether

[0280] ●1uM OGG

[0281] TRM (liquid PAAP preparation)

[0282] 50mM Tris pH 7.5

[0283] 16 mM magnesium chloride

[0284] 100 mM potassium chloride

[0285] ●0.02% triethylene glycol dodecyl ether

[0286] 1.5 mM tris(2-carboxyethyl)phosphine

[0287] 0.4 mg / mL PAAP

[0288] KRM (lyophilized preparation of PAAP)

[0289] 50mM Tris pH 8.8

[0290] 50 mM potassium chloride

[0291] 14.65% trehalose

[0292] 4% (2-hydroxypropyl)-β-cyclodextrin

[0293] 2 mM tris(2-carboxyethyl)phosphine

[0294] ●0.02% triethylene glycol dodecyl ether

[0295] 8uM zinc chloride

[0296] 4 mM MgOAc

[0297] Fig.18 It was shown that there was no difference in performance between conditions using 1 mM and 2 mM magnesium. Furthermore, using KLM2 instead of OLM2 did not affect primary or secondary sequencing metrics.

[0298] Additional Notes

[0299] Although 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 provided herein.

[0300] 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.

[0301] Sequence Listing

[0302] SEQ ID NO.1: Pyrococcus aquatica alkaline phosphatase (PAAP) protein

[0303] MSPSGVRNVIILIGDGMGFSQLQLTKLVYGHLNMEDFPYTGIELTDSLSGE

[0304] VTDSAAAGTAIATGVKTYNRMISTTNVTGKLVNLTTLLEIAQMLGKATG

[0305] LVTTTRITHATPAVFASHVPDRDMEEEIARQLILHNVTVLMGGGREKFSE

[0306] EVLKLAEDYGYSIVYTREDLEKVKDGKVLGLFAEGHLPYVLDRSEEDVS

[0307] LLEMTKKAIEILEKNPNGFFLMIEGGRIDHACHANDVASIVAETKEFDDV

[0308] VGYVLDYARRRGDTLVIVLADHETGGLGIGLNYGHSVDIDSIRRIDASIEE

[0309] MSKEIKSGGDIRDVIRRHTGLELTDEEVKEIEEAKNSTNKYALGNIIGEIIS

[0310] KKLGVGFVSHKHTGEPVPLLAYGPGAENFVGFKHHVDTAKVIAKLMIFG

[0311] DRSISFTIKGVSKIKGDVTGDYRVDERDAYATLMLLLGDLVDTELENIAD

[0312] MDNNGIIDLLDVMAILQASS

[0313] SEQ ID NO.2: Methanococcus jannaschii oxoguanine glycosylase (MjaOGG) protein

[0314] MMLIKKIEELKNSEIKDIIDKRIQEFKSFKNKSNEEWFKELCFCILTANFTA

[0315] EGGIRIQKEIGDGFLTLPREELEEKLKNLGHRFYRKRAEYIVLARRFKNIK

[0316] DIVESFENEKVAREFLVRNIKGIGYKEASHFLRNVGYDDVAIIDRHILREL

[0317] YENNYIDEIPKTLSRRKYLEIENILRDIGEEVNLKLSELDLYIWYLRTGKVL

[0318] K

[0319] SEQ ID NO.3: P5 sequence

[0320] AATGATACGGCGACCACCGAGATCTACAC

[0321] SEQ ID NO.4: P7 sequence

[0322] CAAGCAGAAGACGGCATACGAGAT

[0323] SEQ ID NO.5: P5' sequence (complementary to P5)

[0324] GTGTAGATCTCGGTGGTCGCCGTATCATT

[0325] SEQ ID NO.6: P7' sequence (complementary to P7)

[0326] ATCTCGTATGCCGTCTTCTGCTTG

[0327] SEQ ID NO: 12: Thermostable exonuclease from Thermococcus aquatica

[0328] MGSSHHHHHHSSGLVPRGSHMRIVAADTGGAVLDESLQPIGLISTVAVLV

[0329] EKPYNTAKEFLVKFSDPYNYDLSGRQAILDEINLAIELAKRTKPDVVHLD

[0330] STLGGIEVRKLDEPTIEALSISDKGKEVWKDLSKDVQPLAKKFWEETGIEI

[0331] LAIGKSSVPVRIAEIYSGIFSVKWAIEEVKKGHKDHLIIGLPRYMGVEIRDG

[0332] KITGRTLDPREGNLYGEISIEVPNNIKWEVYPNPLVRRFHIFEIWGEY

[0333] SEQ ID NO:13: Thermostable Exonuclease from Pyrococcus furiosus

[0334] MGSSHHHHHHSSGLVPRGSHMRIVAADTGGAVLDETFEPIGLIATVAVL

[0335] VEKPYRSAKEVMVKYANPYDYDLTGRQAIRDEVLLAIELARKVKPDVIH

[0336] LDSTLGGIELRKLDEPTIDALGISDKGKEVWKELSKDLQPLARKFWEETNI

[0337] EIVAIGKSSVPVRIAEIYAGIYSAKWGIENVEKEGHLIIGLPRYMEVNIKDG

[0338] KIIGRSLDPREGGLYGSAEVSVPEGVKWEIYPNPVARRFMIFEIFSKR。

Claims

1. A resynthesis kit comprising thermophilic phosphatase and polymerase.

2. The resynthesis kit of claim 1, wherein the kit comprises the thermophilic phosphatase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

3. The resynthesis kit according to claim 1 or claim 2, wherein the thermophilic phosphatase is derived from a thermophilic bacterium, wherein the thermophilic bacterium belongs to the genus Thermococcus. 4 . The resynthesis kit according to claim 1 , wherein the thermophilic phosphatase comprises an amino acid sequence defined by SEQ ID NO: 1, or a functional variant or functional fragment thereof.

5. The resynthesis kit according to any one of claims 1 to 4, wherein the thermophilic phosphatase has a temperature of 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, Denaturation temperature of 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, 100°C or higher, 105°C or higher, or 110°C or higher.

6. The resynthesis kit according to any one of claims 1 to 5, wherein the kit comprises the polymerase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM. 7 . The resynthesis kit according to claim 1 , wherein the polymerase is a thermophilic polymerase.

8. The resynthesis kit according to claim 7, wherein the thermophilic polymerase has a denaturation temperature of 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, 65°C or more, 70°C or more, 75°C or more, 80°C or more, 85°C or more, 90°C or more, 95°C or more, 100°C or more, 105°C or more, or 110°C or more.

9. The resynthesis kit according to any one of claims 1 to 8, wherein the kit further comprises a thermophilic glycosylase, preferably a thermophilic oxoguanine glycosylase.

10. The resynthesis kit of claim 9, wherein the kit comprises the thermophilic glycosylase at a concentration of about 0.01 μM to about 1000 μM, about 0.02 μM to about 100 μM, about 0.05 μM to about 50 μM, about 0.1 μM to about 20 μM, or about 0.2 μM to about 10 μM.

11. The resynthesis kit according to claim 9 or claim 10, wherein the thermophilic glycosylase is derived from a thermophilic bacterium, wherein the thermophilic bacterium belongs to the genus Methanococcus. 12 . The resynthesis kit according to claim 9 , wherein the thermophilic glycosylase comprises an amino acid sequence defined by SEQ ID NO: 2, or a functional variant or functional fragment thereof.

13. The resynthesis kit according to any one of claims 9 to 12, wherein the thermophilic glycosylase has a denaturation temperature of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, or 95°C or higher.

14. The resynthesis kit according to any one of claims 1 to 13, wherein the kit further comprises an exonuclease, preferably a thermophilic exonuclease.

15. The resynthesis kit of claim 14, wherein the kit comprises a thermophilic exonuclease at a concentration between about 1 ug / ml and about 100 ug / ml.

16. The resynthesis kit according to claim 14 or claim 15, wherein the thermophilic exonuclease is derived from Thermococcus abyssinus or Thermococcus furiosus. 17 . The resynthesis kit according to claim 16 , wherein the thermophilic exonuclease comprises the amino acid sequence defined by SEQ ID NO: 12, or a functional variant or functional fragment thereof.

18. The resynthesis kit according to claim 16, wherein the thermophilic exonuclease comprises an amino acid sequence defined by SEQ ID NO: 13, or a functional variant or functional fragment thereof.

19. The resynthesis kit according to any one of claims 1 to 18, wherein the thermophilic phosphatase is in a lyophilized formulation.

20. The resynthesis kit according to claim 19, wherein the lyophilized preparation comprises any one or more of the thermophilic phosphatase, a salt, an exonuclease, a detergent, and magnesium chloride, magnesium acetate, or magnesium sulfate.

21. The resynthesis kit of claim 20, wherein the phosphatase comprises Thermococcus aquatica alkaline phosphatase.

22. The resynthesis kit according to any one of claims 9 to 18, wherein the thermophilic oxoguanine glycosylase is in a lyophilized formulation.

23. The resynthesis kit according to claim 22, wherein the lyophilized preparation comprises the thermophilic oxoguanine glycosylase, a salt and trehalose.

24. The resynthesis kit according to any one of claims 1 to 23, wherein the kit comprises at least one selected from the group consisting of a recombinase, a single-stranded nucleotide binding protein, a nucleotide triphosphate (NTP), a substrate for generating ATP, and an enzyme for generating ATP.

25. The resynthesis kit according to any one of claims 1 to 24, wherein the kit comprises a recombinase, a single-stranded nucleotide binding protein, nucleotide triphosphates (NTPs), a substrate for generating ATP, and an enzyme for generating ATP.

26. The resynthesis kit according to any one of claims 1 to 25, wherein the kit further comprises a metal cofactor composition, preferably wherein the metal cofactor composition comprises magnesium ions.

27. The resynthesis kit according to any one of claims 1 to 26, wherein the kit further comprises instructions for using the kit in resynthesis of nucleic acid templates or paired sequencing of nucleic acid templates.

28. Use of the resynthesis kit according to any one of claims 1 to 27 in the resynthesis of nucleic acid templates or paired sequencing of nucleic acid sequences.

29. A method for resynthesizing a nucleic acid sequence, wherein the method comprises: removing a blocking phosphate group from the blocked primer using a thermophilic phosphatase to form an unblocked primer; as well as A polymerase and the first nucleic acid template are used to form a second nucleic acid template that is extended from the deblocked primer.

30. The method according to claim 29, wherein the thermophilic phosphatase is as defined in any one of claims 2 to 5.

31. A method according to claim 29 or claim 30, wherein the polymerase is as defined in any one of claims 6 to 8.

32. The method according to any one of claims 29 to 31, wherein the blocked primer and / or the deblocked primer is immobilized on a solid support, preferably wherein the solid support is a flow cell.

33. The method of any one of claims 29 to 32, wherein the step of forming the second nucleic acid template extended from the deblocked primer is performed using bridge amplification.

34. The method according to any one of claims 29 to 33, wherein the method further comprises the step of isolating the first nucleic acid template by using a thermophilic glycosylase.

35. The method according to claim 34, wherein the thermophilic glycosylase is as defined in any one of claims 9 to 13.

36. The method according to any one of claims 29 to 35, wherein the method is performed isothermally.

37. The method of claim 36, wherein the method is 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.

38. A method for sequencing a nucleic acid sequence by paired sequencing, wherein the method comprises: sequencing the first nucleic acid template; Resynthesis of a nucleic acid sequence using the method according to any one of claims 29 to 37 to form a second nucleic acid template; as well as The second nucleic acid template is sequenced.

39. The method of claim 38, wherein the step of sequencing the first nucleic acid template and / or the step of sequencing the second nucleic acid template is performed using a sequencing-by-synthesis technique or a sequencing-by-ligation technique.

40. The method according to any one of claims 29 to 39, wherein the method comprises using a resynthesis kit according to any one of claims 1 to 27.

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