A double-end sequencing method

Abstract

This invention discloses a paired-end sequencing method, comprising: providing a solid-phase medium with reaction sites on its surface, wherein a double-stranded nucleic acid molecule containing a target nucleic acid sequence is immobilized on the reaction sites, and linearizing the molecule to obtain a first strand for sequencing; providing a new amplification primer to the solid-phase medium and blocking its 3' end; providing a sequencing reaction solution for sequencing the first strand, wherein the substrate nucleotide contained in the sequencing reaction solution is a nucleotide with a 5' end phosphate-fluorescent switching label and a 3' end containing a reversible end-capping termination group; unblocking the aforementioned new amplification primer and amplifying the nucleotide to obtain a double-stranded nucleic acid; linearizing the primer to obtain a second strand for sequencing; and providing a sequencing reaction solution containing the aforementioned substrate nucleotide for sequencing the second strand. The method relies on a substrate nucleotide with a novel structure, where the fluorescent group can be released through enzymatic cleavage, avoiding molecular scarring and facilitating increased sequencing read length. Furthermore, since the fluorescent group is attached to the terminal phosphate, nucleotide substrate synthesis is cheaper.

Description

Technical Field

[0001] This invention belongs to the field of gene sequencing, specifically, it relates to a paired-end sequencing method. Background Technology

[0002] With the completion of the Human Genome Project, scientists began searching for more efficient methods to explore the complexity of the genome. Traditional sequencing technologies, such as Sanger sequencing, while impeccable in read accuracy, have limited throughput, making them unsuitable for large-scale genome research. To overcome the limitations of Sanger sequencing, high-throughput sequencing technologies emerged. High-throughput sequencing can generate massive amounts of data in a short time, greatly advancing research in genomics and related fields. For example, Illumina's Solexa sequencing technology was one of the early high-throughput sequencing technologies. Through nearly two decades of research, high-throughput sequencing technology has made breakthrough progress, with sequencing throughput, accuracy, and read length limits gradually improving. However, current mainstream high-throughput sequencing technologies still have many shortcomings. For instance, molecular scarring left during reversible sequencing termination severely hinders improvements in sequencing accuracy and read length. Paired-end sequencing is a common high-throughput sequencing mode. This mode improves sample utilization, requiring only one library preparation for two sequencing runs; it yields highly accurate sequencing data with longer read lengths and can detect insertion and deletion variations that cannot be detected by single-end sequencing data. However, existing mainstream paired-end sequencing technologies rely on reversible termination sequencing, which leads to problems such as molecular scarring during the sequencing process and limited read lengths. Furthermore, two-stranded sequencing, particularly paired-end sequencing, is often inferior to single-stranded sequencing in terms of both signal strength and accuracy. Therefore, a new paired-end sequencing technology is needed to address these issues. Summary of the Invention

[0003] Specifically, this invention discloses a paired-end sequencing method, characterized by comprising:

[0004] A solid-phase medium is provided, the surface of which contains reaction sites, and the reaction sites are immobilized with a double-stranded nucleic acid molecule containing a target nucleic acid sequence, the double-stranded nucleic acid molecule being obtained by first amplification of the target nucleic acid using amplification primers; the amplification primers contain cleavage sites.

[0005] The double-stranded nucleic acid molecule is subjected to a first treatment to obtain a first strand that is essentially single-stranded for sequencing.

[0006] A new amplification primer is provided to the solid medium, and the 3' end of the new amplification primer is closed.

[0007] A sequencing reaction solution is provided for first-strand sequencing. The sequencing reaction solution contains a substrate nucleotide, which is a nucleotide with a 5' end phosphorylated fluorescently switched label and a 3' end containing a reversible termination group.

[0008] Unblock the new amplification primers and perform a second amplification to obtain double-stranded nucleic acid.

[0009] The nucleic acid double strands are subjected to a second processing to obtain a second strand that is essentially single-stranded for sequencing.

[0010] A sequencing reaction solution is provided for second-strand sequencing. The sequencing reaction solution contains a substrate nucleotide, which is a nucleotide with a 5' end phosphorylated fluorescently switched label and a 3' end containing a reversible termination group.

[0011] The paired-end sequencing of the target nucleic acid was completed by determining the sequences of the first and second strands.

[0012] According to a preferred embodiment, the structure of the substrate nucleotide is as shown in formula (Ⅰ):

[0013]

[0014] Wherein, Y is O or S, B is a heterocyclic base, and n is an integer from 0 to 6; R is selected from azidomethyl, amino, allyl, substituted dithioalkyl, substituted methoxymethyl, o-nitrobenzyl, coumarin, phosphate nitrile ethyl ester, trimethylsilyl, tetrahydropyranyl, azido, alkyl hydroxyamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, or vinyl; Fluorogenic Dye is selected from anthracene, phenoxazine, acridine, or coumarin with fluorescence switching properties, wherein the anthracene with fluorescence switching properties includes one or more of oxanthracene-fluorescein, carbamate-Beijing orange, silanthracene, germananthracene, phosphoxanthracene, or thioanthracene.

[0015] According to a preferred embodiment, the first amplification includes a first amplification primer and a second amplification primer, the first treatment is applied to the first amplification primer, the new amplification primer includes a third amplification primer, and the unblocked third amplification primer contains at least a portion of the consistent sequence of the first amplification primer, the length of the consistent sequence being at least 15 nt, preferably, the length of the consistent sequence being at least 20 nt.

[0016] According to a preferred embodiment, the first amplification includes a first amplification primer and a second amplification primer. The first treatment is applied to the first amplification primer. The new amplification primer includes a third amplification primer and a fourth amplification primer. The unblocked third amplification primer contains at least a partially consistent sequence of the first amplification primer, and the unblocked fourth amplification primer contains at least a partially consistent sequence of the second amplification primer. The length of the consistent sequence is at least 15 nt, preferably at least 20 nt.

[0017] According to a preferred embodiment, the ratio of the number of the third amplification primer to the number of the fourth amplification primer is 1:1 to 5:1, preferably 2:1 to 3:1.

[0018] According to a preferred embodiment, the 3' end of the new amplification primer is blocked by fixing the new amplification primer to the surface of a solid medium and then modifying the 3' end of the primer, including phosphorylation modification or ddNTP modification.

[0019] According to a preferred embodiment, the 3' end of the new amplification primer is closed, meaning that before the new amplification primer is fixed to the surface of the solid medium, the 3' end of the new amplification primer has been modified, and the modification is selected from one or more of phosphorylation modification, ddNTP modification, inter-arm modification, amino modification, or biotin modification.

[0020] According to a preferred embodiment, the new amplification primer contains a cleavage site, and the new amplification primer is unblocked by acting on the cleavage site, and the cleavage site is different from the cleavage site acted on in the second treatment.

[0021] According to a preferred embodiment, the reaction site comprises multiple reaction volumes created by multiple reaction chambers disposed in a solid medium, wherein the double-stranded nucleic acid molecule containing the target nucleic acid sequence is immobilized in the reaction volume; after the sequencing reaction solution is delivered to each reaction volume, each reaction volume may be sealed and / or separated from other reaction volumes on the array; then, the emission signal from the fluorescent switching label can be detected and / or recorded by each reaction volume.

[0022] According to a preferred embodiment, the method further includes removing a reversible termination group from the 3' end of the incorporated nucleotide before the next sequencing cycle, thereby making the nucleotide in an extendable state.

[0023] This invention discloses a pairwise sequencing method that revolutionizes sequencing chemistry. It designs a novel substrate nucleotide with a fluorescent group attached to the terminal phosphate group. This fluorescent group is released through enzymatic cleavage, leaving no residual group and avoiding molecular scarring. The synthesized polynucleotide strand maintains its native conformation, which is beneficial for increasing sequencing read length. Furthermore, because the fluorescent group is attached to the terminal phosphate group, the nucleotide substrate synthesis is cheaper. To ensure compatibility with this novel substrate nucleotide structure, new amplification primers are re-inserted after the first amplification and before the first strand sequencing. This ensures that the new primers can be effectively used for the second amplification, significantly improving the second strand generation efficiency and sequencing quality. Attached Figure Description

[0024] To more clearly illustrate the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments consistent with the present invention. For those skilled in the art, other embodiments can be obtained from these drawings without creative effort.

[0025] Figure 1 A schematic diagram of the paired-end sequencing process in some specific embodiments of the present invention. Detailed Implementation

[0026] definition

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] The use of the term "comprising" is not restrictive. As used herein, whether in transitional phrases or in the body of the claims, the terms "comprising" and "comprising" are to be interpreted in an open-ended sense. That is, the terms should be interpreted synonymously with the phrases "at least having" or "at least including." For example, when used in the context of a process, the term "comprising" means that the process includes at least the listed steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device contains at least the listed features or components, but may also contain additional features or components.

[0029] The term “and / or” means one or all of the listed elements, or any combination of two or more of the listed elements. “Or” is used herein to mean “and / or” unless the context otherwise requires. In some cases, the use of “and / or” does not imply that its use in other cases is intended not to mean “and / or”.

[0030] The terms "preferred" and "ideally" refer to embodiments of the invention that provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, and is not intended to exclude other embodiments from the scope of the invention.

[0031] Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the direction from amino to carboxyl.

[0032] Unless otherwise stated, all headings are for the reader's convenience and should not be used to limit the meaning of the text following them. The headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the entire specification. Therefore, the terms defined below are defined more fully with reference to the entire specification.

[0033] As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Typically, this enzyme begins synthesis at the 3' end of a primer annealed to the polynucleotide template sequence and proceeds toward the 5' end of the template strand. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides. Deoxyribonucleotides can be native nucleotides or modified or labeled nucleotides.

[0034] As used in this invention, "nucleotide" comprises a nitrogenous heterocyclic base, a ribose sugar, and one or more phosphate groups. These are monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., lacking the oxygen present at the 2' position of the hydroxyl group on the ribose. The nitrogenous heterocyclic base may be a purine, deazopurine, or pyrimidine base. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs, such as 7-deazopurine or 7-deazoguanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and their modified derivatives or analogs. The C-1 atom of the deoxyribose is bonded to the N-1 of a pyrimidine or the N-9 of a purine. In some cases, the term "nucleotide" may also cover nucleotide modifications or conjugates.

[0035] As used in this invention, the target nucleic acid is a polynucleotide or nucleotide cluster of any origin and of a length suitable for sequencing-by-synthesis; preferably, the target nucleic acid is immobilized on the surface of a vector.

[0036] As used herein, “incorporation” means the formation of a covalent bond between the nucleotide described herein and the oligonucleotide or polynucleotide. In some such embodiments, a phosphodiester bond is formed between the 3' hydroxyl group of the oligonucleotide or polynucleotide and the 5' phosphate group of the nucleotide described herein. In this application, “extension” and “incorporation” can be interpreted synonymously.

[0037] Nucleotides with 5' terminal phosphorylated fluorescent labeling and 3' end containing a reversible termination group

[0038] The paired-end sequencing method of this invention first uses a 5'-terminal fluorescently switched phosphate labeling and a nucleotide containing a reversible termination group at the 3' end (referred to as a 5'-terminal fluorescently switched reversible termination nucleotide). See CN104910229A for examples of 5'-terminal fluorescently switched phosphate nucleotides. Hydroxyl-containing xanthracene, coumarin, and halogenated fluorescent molecules are introduced as detection tags via polyphosphate at the 5' end of the nucleotide. This nucleotide structure can serve as a substrate for DNA or RNA polymerases, be recognized by the polymerases, and be incorporated into the DNA or RNA chain, releasing a polyphosphate molecule containing a fluorescent group. The fluorescently switched polyphosphate molecule can then be further degraded as a substrate for an enzyme (e.g., alkaline phosphatase) until all phosphate groups are removed from the fluorescent molecule, thereby generating a fluorescent signal. The aforementioned fluorescent molecule is a fluorophore with fluorescent switching properties. In a preferred embodiment, the sequencing method may include using an enzyme to release the fluorophore with fluorescent switching properties, optionally including first using DNA polymerase to release the polyphosphate-substituted fluorophore, and then using a phosphatase to remove the substituted polyphosphate, thereby releasing the fluorophore. Exemplary fluorophores include phenolic dyes such as fluorescein (e.g., methoxyfluorescein), phenoxazine (e.g., halogen), acridine (e.g., DDAO), and coumarins (e.g., 7-hydroxycoumarin, 3-carboxy-7-hydroxycoumarin, 4-acetic acid-7-hydroxycoumarin, 7-hydroxy-4-trifluoromethylcoumarin), Peking Orange (PO), Tokyo Green (TG), and Tokyo Magenta (example structures of the above fluorophores are shown in Table 1). The chemical reactions of fluorescent nucleic acid substrates based on phenolic dyes are relatively simple because the phenolic oxygen is esterified to a phosphate group. For dyes containing amines (e.g., rhodamine and its derivatives, cresol violet, etc.), nucleotide substrates can be introduced by introducing self-cleaving groups. Once DNA polymerase incorporates the labeled nucleotide substrate and cleaves it between the α- and β-phosphate groups, the released fluorophore fluoresces either directly from the nucleotide cleavage or after further enzymatic action by other enzymes (such as alkaline phosphatase). These new fluorescent molecules are then detected using standard fluorescence detection techniques (e.g., total internal reflection fluorescence, epifluorescence, or confocal microscopy). Fluorescence switching refers to a significant change in the fluorescence signal after sequencing compared to before the sequencing reaction; commonly, the fluorescence signal after sequencing is significantly enhanced (or increases) compared to before the sequencing reaction.

[0039] Table 1 Fluorescent Groups

[0040]

[0041]

[0042] Reversible terminators are well-known in the fields of sequencing and nucleic acid synthesis. Examples include commonly used 3'-modified reversible terminating nucleotides (such as oxyamino groups). Those skilled in the art will understand how to attach a suitable protecting group to the 3' position of the ribose sugar to block the interaction between the polymerase and the 3'-OH. The aforementioned protecting group can be directly attached to the 2' position of the ribose sugar, possessing sufficient size or charge to block the interaction at the 3' position; or the protecting group can be directly attached to the 3' position of the ribose sugar. The protecting group attached to the 2' or 3' position can be cleaved (or removed), reverting to an extendable 3'-OH.

[0043] As used herein, the terms “blocking,” “blocking,” or “terminating” refer to the use of a specific group to protect the 3'-OH or 2'-H (or -OH) of a nucleoside or nucleotide, thereby terminating the polymerization of a potential polymerase (e.g., DNA polymerase). The group used for blocking is called a “blocking group,” “blocking group,” “terminating group,” or “protecting group.” If the blocking group can be removed, allowing the nucleotide to revert to an extended state, such blocking is called “reversible blocking,” “reversible blocking,” or “reversible termination.” The group used for reversible termination is called a “reversible termination group,” and the process of removing the reversible termination group, restoring the 3' position of the ribose to an extended 3'-OH, can be called “deprotection.”

[0044] Regarding reversible terminating groups and corresponding deprotecting reagents: The 3' reversible blocking group (R) used in this article can be selected from the following groups: 3'-O-azidomethyl, 3'-O-amino, 3'-O-allyl, 3'-O-substituted dithioalkyl, 3'-O-substituted methoxymethyl, 3'-O-o-nitrobenzyl, 3'-O-coumarin, 3'-O-phosphazene ethyl ester, 3'-O-trimethsilyl, 3'-O-THP, 3'-O-azido, 3'-O-alkylhydroxyamino, 3'-thiophosphate, 3'-O-malonyl, 3'-O-benzyl, 3'-O-acetal, 3'-O-thiocarbamate, 3'-vinyl, etc., 2'-phosphate, 2'-thiophosphate, etc. Other available reversible terminating groups include, for example, those disclosed in international applications WO2014139596A1 and CN201780039312.5. This invention does not particularly limit the type of reversible terminating group; according to the principles of this invention, any 3'-OH or 2'-H (or -OH) group used in the art for reversible blocking can be used as a reversible terminating group in this invention. For example, 3'O reversible terminating groups that can be cleaved by a reducing agent (e.g., phosphine) include, but are not limited to, azidomethyl. 3'O reversible terminating groups that can be cleaved by ultraviolet light include, but are not limited to, nitrobenzyl. 3'O reversible terminating groups that can be cleaved by contact with an aqueous Pd solution include, but are not limited to, allyl. 3'O reversible terminating groups that can be cleaved by acid include, but are not limited to, methoxymethyl. 3'O reversible terminating groups that can be cleaved by contact with a sodium nitrite buffered aqueous solution (pH = 5.5) include, but are not limited to, aminoalkoxy. Correspondingly, commonly used deprotecting agents can be selected from: for example, for 3'-O-thiocarbamates, removal can be achieved under various conditions, such as, non-limiting exemplary conditions including NaIO4 and potassium persulfate; for 3'-O-acetal, cleavage can be achieved by a palladium catalyst; for 3'-O-azido or 3'-O-azidomethyl, the azide group can be converted to an amino group by contacting a phosphine, such as, non-limiting exemplary conditions including phosphine, such as trialkylphosphine, non-limiting examples of which include tris(hydroxymethyl)phosphine (THP), tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THMP), or tris(hydroxyethyl)phosphine (THEP), etc. For 3'-vinyl, various tetrazine substances can be used to deprotect the 3'-vinyl protecting group. The deprotection mechanism of the 3'-vinyl protecting group may be similar to that of simple tetrazines, for example, substituted tetrazines, each of v-tetrazine, as-tetrazine, and / or s-tetrazine can be used for the deprotection of the 3'-vinyl end-capped group. For the 3'-O-allyl group, the cleavage process uses a ligand of metallic palladium and trisodium triphenylphosphine tris(m-sulfonate) to remove the blocking group via a palladium-catalyzed deallylation reaction.For 3'-O-THP, the THP protecting group is stable under alkaline conditions but unstable under mildly acidic conditions, such as by cleaving it with HOAc / THF / H2O (4:2:1) under slight heating. For the 2'-phosphate group, this modifying group can be removed, for example, by enzymatic methods such as phosphatases, exonuclease III, exonuclease IV, polynucleotide kinases, phosphodiesterases, or a combination of phosphodiesterases and phosphatases. For the 2'-thiophosphate group, the blocking group can be removed using phosphodiesterases (e.g., snake venom esterase).

[0045] Amplification primers

[0046] The term "amplification primer" refers to oligonucleotides used in amplification reactions. These primers can consist entirely of natural or modified nucleotides (e.g., 8-oxoguanine, methylated nucleotides, etc.) and may also include necessary non-nucleotide chemical spacers, including units containing disulfide, dihydroxy, azo, or azido groups, spacers, etc.

[0047] Interarm-like modifications

[0048] Spacer modifications, also known as spacer modifications, mostly consist of straight carbon chains or ethylene glycol. In this application, a spacer is introduced into the 3' end of the newly provided amplification primer. When the spacer is modified at the 3' end of the primer, it can block DNA polymerase extension, allowing the primer to remain in an "inert" state when extension is not required. Commonly used spacers include: C3 spacer, C6 spacer, C12 spacer, Spacer 9, Spacer 18, dSpacer, etc.

[0049] cleavage site

[0050] In this invention, the term "cleavage site" has a broad meaning. Any cleavage method that, when applied to this site, results in the cleavage of one strand of a double-stranded nucleic acid molecule, leaving only a essentially single-stranded polynucleotide chain on the solid-phase medium surface after denaturation, can be called a "cleavage site." Commonly used cleavage methods include: photochemical cleavage, suitable chemical cleavage, suitable enzymatic cleavage, ribonucleotide cleavage, cleavage without a base site, enzymatic digestion with endonucleases, and cleavage of hemimethylated DNA.

[0051] Optical shearing

[0052] In this invention, "photoshearing" includes any method that uses light energy to cleave a single strand of the nucleic acid to be tested. The cleavage site can be located within a non-nucleotide chemical spacer unit in the nucleic acid to be tested. The chemical spacer unit includes the PC spacer phosphorimide (4-(4,4'-dimethoxytriphenylmethoxy)butamidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphorimide, available from Glen Research (Sterling, VA, USA), which can be cleaved by exposure to a UV light source. This spacer unit, along with a thiophosphate group that allows attachment to a solid surface, can be attached to the 5' end of the nucleotide single strand using standard techniques for the chemical synthesis of oligonucleotides.

[0053] Chemical shearing

[0054] In this invention, "chemical cleavage" includes any method that utilizes chemical reactions (including but not limited to redox reactions, hydrolysis reactions, enzymatic reactions, etc.) to promote / achieve cleavage of single-stranded polynucleotides. Typically, single-stranded polynucleotides may include one or more non-nucleotide chemical moieties and / or non-natural nucleotides and / or non-natural backbone links to allow the chemical cleavage reaction to proceed.

[0055] Representative chemical cleavage sites can be disulfide bonds, which can be cleaved using chemical reducing agents such as tris(2-carbonylethyl)phosphohydrochloride (TCEP), mercaptoethanol, DTT, and cysteine. Representative chemical cleavage sites can also be peptide bonds, which can be cleaved using enzymes that promote peptide bond hydrolysis, including proteinase K. Representative chemical cleavage sites can be reductive cleavage linkers, including but not limited to units containing azo or azido groups. For linkers containing azo groups, the cleavage reaction can be completed using sodium dithionite solution; the cleaved residue is inert and requires no further capping, making it very convenient. For linkers containing azido groups, TCEP or hydrazine can be used to complete the cleavage reaction. Standard methods for automated chemical DNA synthesis can be used to incorporate units containing azo or azido groups into amplified oligonucleotides. Representatively, the chemical cleavage site can be an oxidized chemical linker group, including one or more diol linkers; cleavage can be performed using any substance that promotes diol cleavage, preferably periodate (e.g., an aqueous solution of sodium periodate) or potassium permanganate; after treatment with a cleaving agent (e.g., periodate) to cleave the diol, the cleavage product can be treated with a "capping agent" to neutralize the reactive substances generated in the cleavage reaction. Suitable capping agents for this purpose include, but are not limited to, ethanolamine, triethylamine, triethanolamine, arginine, lysine, cysteine, etc. In a preferred embodiment, the capping agent (e.g., ethanolamine) can be mixed with the cleaving agent (e.g., periodate) so that the reactive substances are capped upon formation. One or more diol linkers can be incorporated into the amplified oligonucleotide using standard methods for automated chemical DNA synthesis. Representatively, the chemical cleavage site can also be an acid-sensitive linker group, including but not limited to ketals, acetals, diphenylsiloxanes, carbonates, carbamates, etc. The cleavage can be performed using any reactants or reaction systems that promote the hydrolysis of ketals, acetals, carbonates, carbamates, or diphenylsiloxanes. The preferred reaction conditions are an acidic buffer system with a pH of 2–3, at room temperature or 37°C, for 5–30 minutes to efficiently complete the cleavage. The main products after cleavage are linker units with hydroxyl (or amino, for carbamates) at the tail ends. Byproducts include acetone (or other ketones), diphenylsilanol, carbon dioxide, and other inert substances. These byproducts do not participate in subsequent DNA synthesis reactions and do not require further removal.

[0056] Ribonucleotide cleavage

[0057] One or more ribonucleotides are incorporated into the amplification primers, and then a suitable cleavage agent, including ribonuclease, is used to selectively cleave the phosphodiester bond between deoxyribonucleotides and ribonucleotides, thereby cleaving the nucleic acid molecule to be tested into a single-stranded sequencing template at a specific site. For example, the single strand of nucleotide to be cleaved contains one ribonucleotide to provide a cleavage site. The suitable cleavage agent includes, but is not limited to: metal ions, such as rare earth ions are also effective, especially La3+, Tm3+, Yb3+, Lu3+ ions with high activity, or Fe(3) or Cu(3) or exposure to elevated pH, such as treatment with a base such as sodium hydroxide. It is particularly important to note that the suitable cleavage agent cannot cleave the phosphodiester bond between two deoxyribonucleotides under the same conditions. The basic composition of the ribonucleotide is generally not important, but can be selected to optimize chemical (or enzymatic) cleavage. For example, if cleavage is to be performed by exposure to metal ions, especially rare earth metal ions, rUMP or rCMP is generally preferred. For cleavage by ribonuclease, it is preferable to include two or more consecutive ribonucleotides, such as 2–10 or 5–10 consecutive ribonucleotides. The exact sequence of the ribonucleotides is generally not important; suitable RNases include, for example, RNase A, which cleaves after the C and U residues. Therefore, when cleaved with RNase A, the cleavage site must contain at least one C or U ribonucleotide. Amplified oligonucleotides incorporating one or more ribonucleotides can be readily synthesized using standard techniques for oligonucleotide chemical synthesis with suitable ribonucleotide precursors.

[0058] In this invention, a "base-free site" refers to a location in a nucleic acid molecule where the base component has been removed. Base-free sites can naturally exist in DNA under physiological conditions through the hydrolysis of nucleoside residues, and can also be formed chemically under artificial conditions (e.g., through enzymatic action). Once formed, base-free sites can be cleaved using suitable cleavage methods (e.g., by using endonucleases or other single-stranded cleaving enzymes, exposure to heat, or alkali treatment), thereby providing a means for site-specific cleavage and capture of nucleic acids. Those skilled in the art will recognize that the use of heat or alkali may denature the nucleic acid molecule and therefore may not be a preferred embodiment.

[0059] In a preferred embodiment, a base-free site can be generated at a predetermined location on the amplified oligonucleotide by first cleaving it with deoxyuridine (U) at the predetermined cleavage site, followed by removal of the uracil base using uracil DNA glycosylase (UDG), thereby generating the base-free site at the specific location. The strand containing the base-free site can then be cleaved at the base-free site using an endonuclease (e.g., EndoIV endonuclease, AP lyase, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase), heat, or alkali treatment. Besides deoxyuridine, base-free sites can also be generated on non-natural / modified deoxyribonucleotides and cleaved in a similar manner using an endonuclease, heat, or alkali treatment. For example, 8-oxoguanine can be converted to a base-free site by exposure to FPG glycosylase; deoxyinosine can be converted to a base-free site by exposure to AlkA glycosylase; and the resulting base-free site can then typically be cleaved by treatment with a suitable endonuclease (e.g., Endo IV, AP lyase). It is important to note that since non-natural / modified nucleotides will be incorporated into the amplification oligonucleotides for amplification purposes, the non-natural / modified nucleotides in this invention should be suitable for polymerase replication reactions that can be used in amplification reactions. In a preferred embodiment, a suitable glycosylase and one or more suitable endonucleases can be mixed together for the cleavage reaction. In such mixtures, the glycosylase and endonuclease will typically be present in an activity ratio of at least about 2:1. In one specific implementation, the USER reagent, available from New England Biolabs, is used to create a single nucleotide nick at the uracil base in the amplified oligonucleotide. Treatment with an endonuclease produces a 3'-phosphate moiety at the cleavage site, which can be removed by a suitable phosphatase (such as alkaline phosphatase) if necessary.

[0060] Enzymatic digestion with nick endonuclease

[0061] In molecular biology, using nicking endonucleases to cleave one strand of a double-stranded nucleic acid molecule is a commonly used technique. Nicking endonucleases are enzymes that selectively cleave one strand of a double-stranded nucleic acid and are well-known in molecular biology. This method can use virtually any nicking endonuclease, provided that the cleavage site present on the amplified oligonucleotide contains a suitable recognition sequence.

[0062] cleavage of hemimethylated DNA

[0063] Hemimethylated DNA refers to oligonucleotides containing one or more methylated nucleotides that, when paired with unmethylated deoxyribonucleotides on their complementary strands, result in a hemimethylated double-stranded structure upon annealing of the two strands. Cleavage of this nucleic acid can be achieved through site-specific cleavage using endonucleases; endonucleases are specific to recognition sequences containing methylated nucleotides. Amplified oligonucleotides incorporating one or more methylated nucleotides can be prepared using appropriate methylated nucleotide precursors and standard automated DNA synthesis techniques.

[0064] linearization

[0065] In this application, the term "linearization" refers to the selective removal of the complementary strand. For example, by immobilizing one of the amplification primers and making it cleavable from the surface of a solid medium, double-stranded DNA can be converted into single-stranded DNA using heating or chemical denaturation conditions, yielding a single-stranded molecule containing hybridization sites for sequencing primers. This single-stranded molecule can hybridize with sequencing primers to allow sequencing reads of the immobilized template strand (e.g., the first strand).

[0066] End

[0067] End capping is a common step in sequencing reactions. Common end capping refers to the process of transferring a nucleoside with a non-extending 3' group to the 3' end of a solid nucleic acid (e.g., an unextended amplified oligonucleotide) using enzymes such as terminal transferase (TdT). This end cannot continue the reaction, thus reducing the occurrence of side reactions. Terminal transferase (TdT) is a template-independent DNA polymerase that catalyzes the binding of deoxyribonucleotides to the 3' hydroxyl terminus of a DNA molecule. Single-stranded and double-stranded DNA molecules with protruding, recessed, or smooth ends can all serve as substrates for TdT, with tail lengths ranging from 5 to 300 nt. In this application, the end capping reaction is performed before the sequencing reaction. Invention Details

[0069] Paired-end sequencing is a high-throughput DNA sequencing method that provides more information than single-end sequencing by sequencing both ends of a DNA fragment from two directions, helping to improve the accuracy of sequencing data and solve some complex genomic structure problems. However, despite its significant advantages, paired-end sequencing still has some drawbacks. For example, although paired-end sequencing can improve data accuracy, sequencing errors can still occur, especially in the second strand, which often contains more sequencing errors than the first strand, resulting in a higher error rate. Furthermore, existing paired-end sequencing methods often rely on reversible termination reactions. These reactions fluorescently label the substrate nucleotides, and the "molecular scars" left after each chemical excision of the labeled molecules inevitably cause some degree of morphological change in the newly synthesized DNA molecules, preventing the effective binding of polymerase molecules and thus affecting the read length for sequencing applications.

[0070] Based on this, the present invention discloses a novel paired-end sequencing method that can at least partially solve the aforementioned technical problems. Specifically, the paired-end sequencing method of this application relies on a newly designed substrate nucleotide, which is a nucleotide with a 5' terminal phosphate fluorescent switching label and a 3' reversible termination. The advantage of sequencing using this terminally phosphate-labeled substrate nucleotide is that after the polymerase correctly recognizes and reacts with the labeled substrate nucleotide, it cleaves and releases the remaining labeled polyphosphate group. The newly synthesized DNA has no residual groups or molecular fragments, thus ensuring the natural structure of the newly synthesized DNA. Simultaneously, because the 3' end of the substrate nucleotide is reversibly terminated, only one substrate nucleotide is extended per sequencing cycle. Using the 5' terminally phosphate-labeled reversibly terminated nucleotide disclosed in this invention for sequencing reactions, since the fluorescent group is attached to the terminal phosphate and can be released through enzymatic reactions, there are no residual groups, avoiding the formation of molecular scars. The synthesized polynucleotide chain can maintain its native conformation, which is beneficial for increasing sequencing read length. Furthermore, because the fluorescent group is attached to the terminal phosphate, nucleotide substrate synthesis is cheaper.

[0071] Specifically, this application provides a paired-end sequencing method, including:

[0072] A solid-phase medium is provided, the surface of which contains reaction sites, and the reaction sites are immobilized with a double-stranded nucleic acid molecule containing a target nucleic acid sequence, the double-stranded nucleic acid molecule being obtained by first amplification of the target nucleic acid using amplification primers; the amplification primers contain cleavage sites.

[0073] The double-stranded nucleic acid molecule is subjected to a first treatment to obtain a first strand that is essentially single-stranded for sequencing; new amplification primers are provided to the solid-phase medium, and the 3' ends of the new amplification primers are blocked.

[0074] A sequencing reaction solution is provided for first-strand sequencing, wherein the sequencing reaction solution contains a substrate nucleotide, which is a nucleotide with a 5' end phosphorylated fluorescently switched label and a 3' end containing a reversible termination group;

[0075] Unblock the new amplification primers and perform a second amplification to obtain double-stranded nucleic acid.

[0076] The nucleic acid double strands are subjected to a second processing to obtain a second strand that is essentially single-stranded for sequencing.

[0077] A sequencing reaction solution is provided for second-strand sequencing, wherein the sequencing reaction solution contains a substrate nucleotide, which is a nucleotide with a 5' end phosphorylated fluorescently switched label and a 3' end containing a reversible termination group;

[0078] The paired-end sequencing of the target nucleic acid was completed by determining the sequences of the first and second strands.

[0079] Flowcharts illustrating some specific embodiments of the paired-end sequencing method of this invention can be found in [reference needed]. Figure 1Two amplification primers (i.e., the first amplification primer and the second amplification primer) are planted on a solid medium (e.g., a chip), and both primers contain cleavage sites (step (1)). Before the first amplification begins, the single-stranded target nucleic acid template is first hybridized with the amplification primers on the chip surface (step (2)). Then, initial extension (step (3)), unwinding and removal of the liquid template are performed (step (4)). Then, the first amplification of the template is performed (steps (5)(6)). After that, cleavage (step (7)), end capping (step (8)) and unwinding reaction are performed (step (9)). At this time, a new amplification primer (the third amplification primer) is planted on the chip surface. The 3' end of the primer is reversibly blocked (step (10)). At this time, the template on the chip is single-stranded (i.e., the first strand). The first sequencing primer is hybridized to the first strand to perform the first strand sequencing (step (11)). Next, the third amplification primer that was replanted was unblocked to make it extendable (step (12)). Then, the primers on the chip surface were used to amplify the double-stranded molecules for second-strand sequencing (step (13)). Then, shearing solution was added to shear the first strand (step (14)). End-capping solution was added to the chip so that the 3' ends of all solid DNA were sealed (step (15)). The strands were unwound (step (16)). At this time, the single strand on the chip surface was the second strand. Then, the second sequencing primer was hybridized to perform the second-strand sequencing reaction (step (17)). After the above steps, the double-end sequencing of the target nucleic acid was completed.

[0080] In a preferred embodiment, the substrate nucleotide used in the sequencing reaction has the structure of formula (I):

[0081]

[0082] Wherein, Y is O or S, B is a heterocyclic base, and n is an integer from 0 to 6; R is selected from azidomethyl, amino, allyl, substituted dithioalkyl, substituted methoxymethyl, o-nitrobenzyl, coumarin, phosphate nitrile ethyl ester, trimethylsilyl, tetrahydropyranyl, azido, alkyl hydroxyamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, and vinyl; Fluorogenic Dye is selected from anthracene, phenoxazine, acridine, and coumarin classes with fluorescence switching properties. The anthracene classes with fluorescence switching properties include one or more of oxanthracene-fluorescein, carbamate-Beijing orange, silanthracene, germananthracene, phosphosoxanthracene, or thioanthracene. This substrate nucleotide has special advantages: because the fluorescent group is attached to the terminal phosphate group, and the fluorescent group can be released through enzymatic reactions, there is no group residue, avoiding the formation of molecular scars. The synthesized polynucleotide chain can maintain its native conformation, which is beneficial for increasing sequencing read length. Furthermore, because the fluorescent group is attached to the terminal phosphate group, nucleotide substrate synthesis is cheaper. Moreover, due to the reversible termination at the 3' end, only one substrate nucleotide is extended per sequencing cycle, which helps improve sequencing accuracy.

[0083] In specific embodiments, the first amplification reaction amplifies the target nucleic acid using a first amplification primer and a second amplification primer. Generally, the optical signal of a single molecule is difficult to detect, even by highly sensitive CCDs. Therefore, it is necessary to replicate the fragment to be sequenced multiple times using amplification methods. The amplification methods for target nucleic acids are well known in the art. As used herein, the term "amplification" refers to the process of linearly or exponentially generating amplicon nucleic acid with the same or substantially the same nucleotide sequence as the target nucleic acid. In some embodiments, the amplification reaction includes a suitable thermostable polymerase. In some embodiments, the term "amplification" refers to a method including polymerase chain reaction (PCR). Amplification conditions are known and typically include at least a suitable polymerase, a suitable template, suitable primers or primer sets, suitable nucleotides (e.g., dNTPs), a suitable buffer solution, and suitable annealing application, hybridization and / or extension time and temperature. In some embodiments, the amplification product (e.g., an amplicon) may contain one or more additional and / or different nucleotides compared to the template sequence or a portion thereof that generates the amplicon (e.g., primers may contain "additional" nucleotides, such as a 5' portion that does not hybridize with the template, or one or more mismatched bases within the primer hybridization portion). Nucleic acid amplification can be performed by thermal cycling or isothermal amplification. In some embodiments, amplification occurs on a solid support (e.g., within a flow cell on a chip), where nucleic acids, nucleic acid libraries, or portions thereof are immobilized by primers. In some sequencing methods, nucleic acid libraries are added to a flow cell and immobilized by hybridization with primers under suitable conditions; this type of nucleic acid amplification is commonly referred to as solid-phase amplification. In some embodiments of solid-phase amplification, all or part of the amplification product is synthesized by extension starting from immobilized primers. Solid-phase amplification reactions are similar to standard liquid-phase amplification, except that at least one amplification primer is immobilized on a solid support. In some embodiments, solid-phase amplification comprises only one type of oligonucleotide primer immobilized to a surface or substrate. In some embodiments, solid-phase amplification comprises at least two different types of immobilized primers. In some embodiments, solid-phase amplification may include one type of oligonucleotide primer immobilized on a solid surface and a second type of different oligonucleotide primer in solution. A variety of different types of immobilized or solution-based primers can be used. Non-limiting examples of solid-phase nucleic acid amplification reactions include bridging amplification, emulsion PCR, WildFire amplification, RPA amplification, RAA amplification, and combinations thereof. In some embodiments, rolling circle amplification (RCA) can also be used to amplify nucleic acids. Those skilled in the art will recognize that other methods and techniques for amplifying nucleic acids can also be used in combination with the methods described herein. The embodiments described herein do not specifically limit the DNA amplification method. Preferably, a solid-phase amplification method comprising at least two different solid-phase primers is used.Nucleic acid fragments are replicated multiple times through amplification, allowing the reaction signals corresponding to nucleotide elongation to be detected by devices such as CCD and TDI linear array cameras during the sequencing chemical reaction.

[0084] In a specific implementation, amplification primers are re-provided to the solid-phase medium before the second amplification reaction. Conventional paired-end sequencing methods do not provide new amplification primers before the second amplification reaction, which has significant drawbacks: during the first-strand sequencing process, after dozens or even hundreds of sequencing reaction cycles, the amplification primers remaining on the medium surface inevitably suffer from loss, non-specific binding with proteins such as polymerases, and non-specific extension due to unsuccessful end-capping. All of these factors greatly reduce the amplification primers available for second-strand generation, severely affecting the generation efficiency of the second strand, reducing the sequencing signal of the second strand, and ultimately decreasing the accuracy of second-strand sequencing. Therefore, the implementation method of this application, which uses new amplification primers in the second amplification reaction, is beneficial in increasing the generation efficiency of the second strand, increasing the sequencing signal of the second strand, and thus improving the accuracy of second-strand sequencing. Furthermore, existing technologies also disclose a technique for providing an additional "dormant" primer on the chip surface before the first amplification reaction. However, this technique has several drawbacks. For example, similar to the aforementioned, during the first-strand sequencing process, after dozens or even hundreds of sequencing reaction cycles, the "dormant" primer on the medium surface will inevitably be lost, non-specifically bind to proteins such as polymerases, and undergo non-specific extension due to unsuccessful end capping, thereby reducing the efficiency of second-strand amplification. In addition, since this "dormant" primer is already present on the chip surface before the first amplification reaction, these "dormant" primers may also bind to the template nucleic acid during the first amplification reaction. However, these primers cannot extend, competitively inhibiting the amplification reaction of normal primers, greatly reducing the efficiency of the first amplification reaction, reducing the generation efficiency of the first strand, and thus affecting the signal and accuracy of the first-strand sequencing.

[0085] In a preferred embodiment, new amplification primers are provided to the solid-phase medium before the first-strand sequencing reaction. These new primers must meet specific requirements, namely, blocking their 3' ends so that they cannot extend during first-strand sequencing. For example, one possible way to block the 3' ends is to include modifications at the 3' ends of the amplification primers during primer synthesis. These modifications could include phosphorylation, ddNTP modification, spacer modification, amino modification, biotin modification, or one or more of the aforementioned modifications. The modifications in this application only need to achieve the blocking effect to prevent DNA polymerase extension; the specific type of modification is not limited. Alternatively, primers without 3' ends can be synthesized during primer synthesis. After the amplification primers are immobilized on the chip surface, the 3' ends of the primers are modified. For example, ddNTPs can be added to the 3' ends of the primers via a terminal transferase-mediated sealing reaction. Alternatively, phosphorylation of the 3' ends of the primers can also prevent DNA polymerase-catalyzed DNA strand extension. Such primers do not require special modification of the 3' ends during synthesis, thus significantly reducing synthesis costs.

[0086] In a specific implementation, the 5' end of the re-provided amplification primers contains a specific chemical modification, which immobilizes the primers on the solid-phase medium surface through a reaction with a chemically modified group. For example, the 5' end of the primers contains an alkyne or cycloalkyne (e.g., DBCO) modification, and the solid-phase medium surface contains an azide group modification. Through a click chemical reaction between the alkyne / cycloalkyne and the azide group, the re-provided amplification primers are immobilized on the chip surface for the generation of sequencing templates before the second-strand sequencing reaction. Optionally, the 5' end of the primers contains a thiol modification, and the solid-phase medium surface contains disulfide bonds. The re-provided amplification primers are immobilized on the chip surface through a reaction between the thiol and the disulfide bond.

[0087] The first point to emphasize in this embodiment is that the re-providing of amplification primers must be performed before the first-strand sequencing reaction. For example, primers can be re-provided after the cleavage reaction or after the capping reaction. This is determined by the structure of the substrate nucleotides in the sequencing reaction and the chemical modifications on the chip surface. If the 3' end reversible termination group of the substrate nucleotide is an azidomethyl group, this group needs to be uncapped after each sequencing cycle. However, the chip surface is modified with azido groups. Once the sequencing reaction begins, the uncapping reagent will destroy the azido group modification on the chip surface while removing the 3' azidomethyl group of the substrate nucleotide. As a result, new amplification primers cannot be replanted on the chip surface. Therefore, the planting of new amplification primers must be completed before the first-strand sequencing reaction.

[0088] The second point that needs to be emphasized in this embodiment is that the 3' end of the replanted amplification primer needs to be closed in the initial state, that is, it cannot extend freely. The reason is that if the 3' end of the new primer is open, the primer may extend the substrate nucleotides during one-strand sequencing, generating noise signals that interfere with the normal sequencing signal.

[0089] In a specific implementation, before the second amplification reaction, the previously 3'-blocked new amplification primers need to be unblocked to restore their free extension state for the amplification reaction. The new amplification primers contain cleavage sites, and the unblocking is achieved by acting on these cleavage sites. These cleavage sites are different from the cleavage sites acted on before the second-strand sequencing reaction, and the cleavage conditions are also different. This allows for controlled and advantageous cleavage by selecting different cleavage conditions according to specific needs. For example, if the cleavage site acted on before the second-strand sequencing reaction is uracil, then the cleavage site in the new amplification primers could be 8-oxoguanine; or, the former is 8-oxoguanine, and the latter is uracil; and so on. Preferably, the cleavage site in the new amplification primers is located close to its 3' end, so that the remaining primer length after the cleavage reaction is sufficient for the second amplification, thereby ensuring the efficiency of the second amplification.

[0090] In a specific implementation, the first amplification includes a first amplification primer and a second amplification primer. Before the first-strand sequencing reaction, the nucleic acid strand is broken by cleavage sites on the first amplification primer to achieve linearization. The newly provided amplification primer is the third amplification primer, and the unblocked third amplification primer contains at least a partially consistent sequence of the first amplification primer. Here, "unblocked third amplification primer" refers to the primer that remains attached to the chip surface after linearization. The sequence of the newly provided amplification primer needs to maintain a high degree of consistency with the original first amplification primer, containing at least 15 nt of consistent sequence, preferably at least 20 nt. Since the first amplification primer is broken by cleavage before the first-strand sequencing, it is necessary to provide a primer containing the sequence of the first amplification primer, which in this implementation is the third amplification primer. Ensuring that the newly provided amplification primer has at least 15 nt of consistent sequence with the original amplification primer is beneficial to improving the generation efficiency of the second amplification reaction and increasing the second-strand sequencing signal.

[0091] In a preferred embodiment, the first amplification includes a first amplification primer and a second amplification primer. Linearization is achieved before the first-strand sequencing reaction by acting on the cleavage site of the first amplification primer. The aforementioned new amplification primer includes a third amplification primer and a fourth amplification primer. The unblocked third amplification primer contains at least a partially consistent sequence of the first amplification primer, and the unblocked fourth amplification primer contains at least a partially consistent sequence of the second amplification primer. That is, the re-provided amplification primers need to maintain a high degree of sequence consistency with the original amplification primers, containing at least 15 nt of consistent sequence, preferably at least 20 nt. The applicant surprisingly discovered during experiments that the newly provided amplification primers, in addition to containing the first amplification primer sequence, also contain the second amplification primer sequence (i.e., the fourth amplification primer). Compared to providing only the third amplification primer, this significantly increases the number of second-stranded molecules generated, increases the second-stranded sequencing signal, and helps improve the accuracy of second-stranded sequencing.

[0092] In a preferred embodiment, the ratio of the re-provided third amplification primer to the fourth amplification primer is 1:1 to 5:1, preferably 2:1 to 3:1. Multiple experiments have shown that by adjusting the ratio of the two re-provided amplification primers, the amount of second-strand primer generated can be controlled, increasing the sequencing signal of the second strand and thus improving sequencing accuracy. When the ratio of the re-provided third amplification primer to the fourth amplification primer is 2:1 to 3:1, the sequencing signal of the second strand is significantly improved, and the sequencing accuracy is also improved. When the ratio is 3:1, the sequencing signal of the second strand is the highest, and the sequencing accuracy is also the highest.

[0093] In a preferred embodiment, the efficiency of the second amplification reaction is significantly improved. When using bridge PCR for amplification, the number of reaction cycles is less than or equal to 10, preferably less than or equal to 8, and more preferably less than or equal to 6. Compared with the prior art, the efficiency of double-strand generation in this embodiment is significantly improved, thus significantly reducing the number of amplification cycles required. Under normal circumstances, only no more than 6 cycles are needed to generate enough double-stranded template to obtain a sufficiently high double-stranded sequencing signal.

[0094] In any embodiment of the method described in this invention, the target nucleic acid may be immobilized to a solid-phase medium. In some such embodiments, the solid-phase medium comprises a plurality of immobilized target nucleic acids. Primer polynucleotides hybridize with at least a portion of the target nucleic acids. In some such embodiments, primer polynucleotides hybridize with at least a portion of the target nucleic acids to form primer polynucleotide / target nucleic acid complexes. The solid-phase medium may comprise clusters of primer polynucleotide / target nucleic acid complexes. In some embodiments, the solid-phase medium comprises a flow cell, such as a patterned flow cell comprising a plurality of micro / nanoscale microreaction chambers, each microreaction chamber being separated from each other.

[0095] In any embodiment of the invention disclosed herein, the sequencing reaction of the target nucleic acid can be performed on an array, such as a chip. The array may include, for example, multiple reaction volumes created by multiple reaction chambers disposed on the array. The target nucleic acid sequence or fragments thereof may be immobilized in the reaction volumes, such as by adsorption or specific binding to trap molecules on a solid-phase medium in each reaction volume. After the sequencing reaction solution is provided and delivered to each reaction volume, each reaction volume may be sealed and / or separated from other reaction volumes on the array. Signals such as fluorescence information can then be detected and / or recorded by each reaction volume.

[0096] As used herein, the term "array" refers to a group of sites that can be distinguished from each other based on their relative positions. Different molecules located at different sites in an array can be distinguished from each other based on the position of the site within the array. A single site in an array can include one or more specific types of molecules. For example, a site can contain a single target nucleic acid molecule having a specific sequence, or a site can contain several target nucleic acid molecules having the same sequence (and / or its complementary sequence). Array sites can be defined by features on a substrate or device. Exemplary features include pores, beads (or other particles), protrusions from a surface, ridges on a surface, patterned coatings on a surface, or channels in a surface. For example, each site in an array can be defined by a pore.

[0097] An exemplary structure of the chip is described in application CN108070525A, the contents of which are incorporated herein by reference in their entirety.

[0098] In various embodiments of the present invention, it is necessary to confine the fluorescent molecules released by the sequencing reaction within the microreaction chamber to prevent or reduce fluorophore diffusion and solution evaporation. For example, the microreaction chamber can be sealed with a fluid immiscible with water. Such fluids can be oils or gases immiscible with water. Examples of such oils include mineral oils, silicone oils, fluorinated oils (e.g., perfluorocarbons and HFE-7500, 2-trifluoromethyl-3-ethoxydodecylfluorohexane, or Fluorinert), or hydrocarbon oils (e.g., isoparaffins); examples of such gases include air, nitrogen, oxygen, saturated water vapor, or rare gases.

[0099] In one embodiment, the microreactor can be sealed with fluorinated oil. First, the desired sequencing aqueous phase liquid is introduced into the flow cell, and then fluorinated oil is introduced into the flow cell to seal the sequencing aqueous phase liquid within the microreactor and cover it, thereby preventing the components within the microreactor from diffusing or evaporating.

[0100] In a specific embodiment of the present invention, the sequencing reaction of the first strand or the second strand comprises at least 50 cycles, preferably at least 75, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350, or at least 400, or at least 450, or at least 500, or at least 600, or at least 700, or at least 800, or at least 900, or at least 1000 cycles.

[0101] In a specific embodiment of the invention, the reversible termination group at the 3' end of the incorporated nucleotide is removed before the next sequencing cycle, so that the nucleotide becomes an extendable state.

[0102] Unless otherwise specified, all figures representing amounts of components, molecular weights, etc., as used in this specification and claims should in all cases be understood to be modified by the term "about". Therefore, unless stated to the contrary, the numerical parameters listed in this specification and claims are approximations, which may vary depending on the desired properties sought to be obtained according to the invention. At a minimum, each numerical parameter should be interpreted based on the number of significant digits reported and by applying conventional rounding. Although the numerical ranges and parameters illustrating the broad scope of the invention are approximations, the values ​​listed in specific embodiments are reported as precisely as possible. However, all numerical values ​​inherently include a range, which is necessarily caused by the standard deviation present in their respective test measurements.

[0103] Example 1

[0104] This embodiment describes the synthesis process of a 5'-terminal labeled 3'-azidomethyl-2'-deoxynucleoside derivative that participates as a polymerase substrate in the extension reaction during sequencing. It should be understood that this embodiment only illustrates the general synthetic pathway for the relevant derivatives and does not represent a limitation to the methods shown.

[0105] Part 1: Synthesis of 5'-terminal phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside tetraphosphate. The synthesis of the labeled monophosphate involves first phosphorylating the hydroxyl groups of the molecule to be labeled, as described below:

[0106]

[0107] In the formula,

[0108] R4 is: H, Me, MeO, COOH, SO3H, F, Cl;

[0109] R5 is: H, Me, MeO, COOH, SO3H, F, Cl;

[0110] R6 is: Me, Et, MeO, EtO.

[0111] Add the labeled molecule (Ar-OH, 5 mmole) to 200 mL of anhydrous acetonitrile. Place the solid suspension under argon protection in an ice-water bath for 5 min. Add phosphorus oxychloride (5 eq) to the solution with rapid stirring and stir rapidly for 5 min. Continue to add tributylamine solution (10 eq) to the solution and keep the reaction under ice-water bath stirring for 4 hours. TLC was used to monitor the disappearance of the reactants. After the reactants had largely disappeared, 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the solution. The mixture was stirred in an ice-water bath for 1 hour. The resulting mixture was concentrated to approximately 1 / 4 of its total volume using a rotary evaporator. The solution was then filtered through a 0.22 μm aqueous filter membrane and purified by medium-pressure preparative chromatography and a C18 column (0-50% B gradient, where A = 50 mM TEAA pH = 6.9, B = acetonitrile). The target fraction was collected and concentrated to near dryness using a rotary evaporator. A small amount of purified water was added, and the pH of the solution was adjusted to slightly alkaline (pH = 8) using tetrabutylammonium hydroxide (40% w / v) solution. After freeze-drying, the corresponding ammonium monophosphate solid product was obtained. Some representative molecular information is as follows:

[0112]

[0113]

[0114] 2) Synthesis of 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt: Commercially available 3'-azidomethyl-2'-deoxynucleoside monophosphate was activated and then reacted with pyrophosphate to obtain the corresponding 3'-azidomethyl-2'-deoxynucleoside triphosphate.

[0115]

[0116] Preparation of 3'-azidomethyl-2'-deoxynucleoside monophosphate ammonium salt: Commercially available 3'-azidomethyl-2'-deoxynucleoside monophosphate (5 mmol / L) (if in sodium phosphate form, the sodium monophosphate needs to be ion-exchanged to phosphate form before this step) was mixed with 200 mL of purified water. The pH of the solution was adjusted to slightly alkaline (pH = 8) with tetrabutylammonium hydroxide (40% w / v) solution, at which point the solution changed from turbid to clear. The adjusted solution was then transferred to a 1 L round-bottom flask and freeze-dried to obtain a white solid of 3'-azidomethyl-2'-deoxynucleoside monophosphate tetrabutylammonium salt. The ammonium salt described in this patent is not limited to tetrabutylammonium salt; tributylammonium salt and triethylammonium salt are also suitable options.

[0117] Preparation of 3'-azidomethyl-2'-deoxynucleoside triphosphate: 5 mmol of solid 3'-azidomethyl-2'-deoxynucleoside monophosphate ammonium salt was dissolved in 100 mL of dry acetonitrile. The solvent was removed by rotary evaporation and the solution was then dried under vacuum. This process was repeated twice with the same volume of acetonitrile added and rotary evaporated twice, followed by drying under vacuum for 2 h. 250 mL of dry anhydrous acetonitrile was added to the flask, and after cooling in an ice-water bath, 2 eq of benzylsulfonic acid imidazole salt activator and 3 eq of diisopropylethylamine solution were added. The mixture was stirred in an ice-water bath for 30 min. Ammonium pyrophosphate salt (5 eq) and anhydrous dry magnesium chloride (5 eq) were added to the activated nucleoside monophosphate solution. The mixture was allowed to rise freely to room temperature with stirring for 24 h. The reaction solution was evaporated using a rotary evaporator to remove most of the acetonitrile. 100 mL of 100 mM TEAA buffer (pH = 8.0) was added to the remaining solution, and the mixture was stirred in an ice-water bath for 1 hour. The resulting mixture was extracted twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was removed by separation, and the resulting aqueous phase was filtered through a 0.22 μm aqueous filter membrane. Purification was performed by preparative chromatography using a C18 column (0-30% B for 20 min, where A = 50 mM TEAA pH = 8.0, B = acetonitrile). The target fraction was collected and concentrated to near dryness using a rotary evaporator. The fraction was then evaporated three times (50 mL acetonitrile / evaporation) using dry acetonitrile to effectively remove residual water. The resulting triphosphate product was dissolved in 200 mL of dry acetonitrile, and its concentration was determined by HPLC before being used directly in the next step. (Yield 45%)

[0118] Add CDI activator (5 eq) and diisopropylethylamine (3 eq) to the dissolved 3'-azidomethyl-2'-deoxynucleoside triphosphate acetonitrile solution, and stir at room temperature for 3 h to generate an activated trimetaphosphate nucleoside derivative intermediate. Add methanol (5 eq) to this reaction solution using a microsyringe, stir for 30 min, and then cool in an ice-water bath. Add the previously synthesized labeled monophosphate solid (3 eq) and anhydrous magnesium bromide (5 eq, added at low temperature, exothermic). Continue to cool to room temperature for 40 h. Add 100 mL of 100 mM TEAA buffer solution (pH = 8.0) to this reaction solution, and stir in an ice-water bath for 1 h. Extract the resulting mixed solution twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was removed by separation, and the obtained aqueous phase was filtered through a 0.22 μm aqueous phase filter membrane. It was then purified by high-pressure preparative chromatography and C18 column chromatography (0-20% B for 20 min, 20-70% B for 15 min, where A is 50 mM TEAA solution, pH=8.0, B=acetonitrile). The target fraction with a purity ≥99% was analyzed and collected (the impure part was collected, concentrated and purified again). The fractions that met the purity requirements were collected and concentrated by rotary evaporator, redissolved in 100 mL of purified water, and then freeze-dried to obtain the target product.

[0119] Part 2: Synthesis of 5'-terminal phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside pentaphosphate

[0120] 1) Preparation of ammonium trimetaphosphate: 20g of commercial sodium trimetaphosphate was dissolved in water and exchanged with Dowex 50W X8(H) ion exchange resin to obtain a trimetaphosphate solution. The pH of the solution was adjusted to slightly alkaline (pH=8.0) with tetrabutylammonium hydroxide (40% W / V) solution. After adjustment, the solution was transferred to a 1L round bottom flask and freeze-dried to obtain a white solid of tetrabutylammonium trimetaphosphate.

[0121] 2) Preparation of the labeled ammonium monophosphate: The labeled monophosphate obtained according to the method described in Part 1 of Example 1 was added to 200 mL of anhydrous acetonitrile. The solid suspension was placed under argon protection and kept in an ice-water bath for 5 min. Phosphorus oxychloride (5 eq) was added to the solution under rapid stirring and stirred rapidly for 5 min. Tributylamine solution (10 eq) was added to the solution and the reaction was carried out under ice-water bath stirring for 4 hours. TLC was used to monitor the disappearance of the reaction raw materials. After the raw materials had basically disappeared, 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the solution. The mixture was stirred and reacted for 1 hour under an ice-water bath. The resulting mixed solution was concentrated to about 1 / 4 of the total volume using a rotary evaporator. The solution was filtered through a 0.22 μm aqueous filter membrane and purified by medium-pressure preparative chromatography and a C18 column (0-50% B gradient, where A = 50 mM TEAA pH = 6.9, B = acetonitrile). The target fraction was collected and concentrated to near dryness using a rotary evaporator. A small amount of purified water was added, and the pH of the solution was adjusted to slightly alkaline (pH = 8.0) using tetrabutylammonium hydroxide (40% W / V) solution. After freeze-drying, the corresponding ammonium monophosphate solid product was obtained.

[0122] 3) Synthesis of 5'-terminal phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside pentaphosphate:

[0123] 2 mmol of solid ammonium trimetaphosphate was placed in a 500 mL round-bottom flask, and 50 mL of anhydrous dry acetonitrile was added. The solvent was removed by rotary evaporation and the mixture was then dried under vacuum. The same volume of acetonitrile was added and rotary evaporated twice, followed by drying under vacuum for 2 h. 250 mL of dry anhydrous acetonitrile was added to the flask, and after cooling in an ice-water bath, 1.5 eq of benzylsulfonic acid imidazole salt activator and 3 eq of diisopropylethylamine solution were added. The mixture was stirred in an ice-water bath for 30 min. 1 eq of dried labeled ammonium monophosphate was added to the activated trimetaphosphate solution, and the mixture was kept in an ice-water bath for 3 h. 1 eq of dried 3'-azidomethyl-2'-deoxynucleoside ammonium monophosphate and 5 eq of anhydrous dry magnesium chloride were then added to the solution. The mixture was allowed to rise freely to room temperature for 24 h with stirring. The reaction solution was removed by rotary evaporation to remove most of the acetonitrile. 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution, and the mixture was stirred in an ice-water bath for 1 hour. The resulting mixture was extracted twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was removed by separation, and the resulting aqueous phase was filtered through a 0.22 μm aqueous filter membrane. Purification was performed by preparative chromatography and a C18 column (0-30% B for 20 min, where A = 50 mM TEAA pH = 8.0, B = acetonitrile). The fractions with ≥99% purity were collected and analyzed (impure fractions were collected, concentrated, and purified again). Fractions meeting the purity requirements were concentrated by rotary evaporation, redissolved in 100 mL of purified water, and freeze-dried to obtain the target product.

[0124] Part 3: Synthesis of 5'-terminal phosphorylated fluorescently labeled 3'-azidomethyl-2'-deoxyribonucleophosphate

[0125] 1) Preparation of ammonium trimetaphosphate: 20g of commercial sodium trimetaphosphate was dissolved in water and exchanged with Dowex 50W X8(H) ion exchange resin to obtain a trimetaphosphate solution. The pH of the solution was adjusted to slightly alkaline (pH=8.0) with tetrabutylammonium hydroxide (40% W / V) solution. After adjustment, the solution was transferred to a 1L round bottom flask and freeze-dried to obtain a white solid of tetrabutylammonium trimetaphosphate.

[0126] 2) Preparation of 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt: The 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt obtained in accordance with the method described in Part 1 of Example 1 was prepared.

[0127] 3) Synthesis of 5'-terminal phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside hexaphosphate: 2 mmol of solid ammonium trimetaphosphate was placed in a 500 mL round-bottom flask, and 50 mL of anhydrous dry acetonitrile was added. The solvent was removed by rotary evaporation and the mixture was then dried under vacuum. The same volume of acetonitrile was added and rotary evaporated twice, followed by drying under vacuum for 2 h. 250 mL of dry anhydrous acetonitrile was added to the flask, and after cooling in an ice-water bath, 1.5 eq of benzylsulfonic acid imidazole salt activator and 3 eq of diisopropylethylamine solution were added. The mixture was stirred in an ice-water bath for 30 min. 1 eq of dried ammonium trimetaphosphate was added to the activated trimetaphosphate solution, and the reaction was maintained in an ice-water bath for 3 h. Then, 1 eq of dried 3'-azidomethyl-2'-deoxynucleoside ammonium triphosphate and 5 eq of anhydrous dry magnesium chloride were added to the solution. The mixture was allowed to rise freely to room temperature for 24 h with stirring. The reaction solution was removed by rotary evaporation to remove most of the acetonitrile. 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution, and the mixture was stirred in an ice-water bath for 1 hour. The resulting mixture was extracted twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was removed by separation, and the resulting aqueous phase was filtered through a 0.22 μm aqueous filter membrane. Purification was performed by preparative chromatography and a C18 column (0-30% B for 20 min, where A = 50 mM TEAA, pH = 8.0, B = acetonitrile). The fractions with ≥99% purity were collected and analyzed (impure fractions were collected, concentrated, and purified again). Fractions meeting the purity requirements were concentrated by rotary evaporation, redissolved in 100 mL of purified water, and freeze-dried to obtain the target product solid.

[0128] Example 2

[0129] 1. Inject a hydrophobic modification reagent into the encapsulated chip cavity, react on the inner surface of the cavity to form a hydrophobic film, and wash away excess unreacted reagent.

[0130] 2. Inject the hydrogel polymerization precursor reagent into the chip cavity, inject the oil phase reagent to seal the precursor in the micro pit, heat and react for a period of time to form a hydrogel in the micro pit, and wash away the excess unreacted reagent.

[0131] 3. Inject a group conversion reagent into the chip cavity to convert the amino groups on the hydrogel into azide groups, and wash away any excess unreacted reagent.

[0132] 4. Inject diphenylcyclooctyne (DBCO) primer reagent into the chip cavity, and use a click reaction to seed the primers onto the hydrogel surface. Wash away any excess unreacted reagent. The seeded primers include a first amplification primer and a second amplification primer, wherein the first amplification primer contains a dU cleavage site and the second amplification primer contains an 8-oxoguanine cleavage site.

[0133] 5. Template prehybridization: Based on the final template concentration added in the amplification reaction, further dilute the template and add an appropriate volume of 0.1M NaOH to unwind it. After adding 0.1M NaOH, shake to mix, centrifuge quickly for 2 seconds, and let it stand at room temperature for 5 minutes.

[0134] After unwinding, add 5x SSC solution to a final volume of 400 μL. Mix gently by pipetting 10 times and immediately place on ice. Do not vortex. Take the amplification chip and wash with 500 μL of SEQ buffer. Add template solution to the chip, adding 200 μL of reaction solution to each chip. Seal with sealing film and place in a plate PCR instrument. Select the anneal program: 96℃, 30 s; -0.05℃ / s; 40℃, 10 s; 25℃, forever. After the reaction is complete, remove and place on ice.

[0135] 6. Initial extension reaction

[0136] 1) Take 400ul of 2X phusion mixture and place it on ice. Then add 400ul of ultrapure water to dilute it by half. Shake to mix well and place it on ice.

[0137] 2) Add the above mixture to the chip, 400 μL per chip, and place the chip on a plate PCR instrument and heat at 72°C for 2 min.

[0138] 3) After the reaction is complete, add 400 μL of formamide into the chip, react at room temperature for 5 minutes, and then clean the chip with cleaning solution.

[0139] 7. Recombinase polymerase reaction

[0140] 1) Prepare the reaction solution according to the table below.

[0141] reagents V / ul hydration solution 180 Magnesium acetate 15 Ultrapure water 95.25 stabilizer 7.5 Total volume 300

[0142] 2) After preparation, shake to mix well and centrifuge quickly for 2 seconds.

[0143] 3) Clean the chip with 500ul of cleaning solution. If there are bubbles, add 200ul of isopropanol to remove the bubbles before cleaning with 500ul of cleaning solution.

[0144] 4) Add the prepared amplification reaction solution to the chip, place the chip on the PCR instrument, tighten the cap, and select the heating program: 40℃, 60min; 4℃, forever.

[0145] 8. The shearing buffer (USER enzyme mix) reacts with the first amplification primer.

[0146] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the U-cutting reaction solution according to the table below.

[0147] reagents V / ul USER enzyme 4 Cutsmart 40 Ultrapure water 346 stabilizer 10 Total 400

[0148] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0149] 3) Clean the chip after the amplification reaction with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0150] 4) Add the prepared shearing reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the shearing reaction program: 37℃, 30min; 4℃, forever.

[0151] 9. Unwinding

[0152] 1) Clean the chip after the shearing reaction with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0153] 2) Add 200 μL of formamide to each chip and react at room temperature (25°C) for 10 min.

[0154] 3) After the reaction is complete, add 1 ml of 1xTE for washing. If sequencing is not performed immediately, seal with sealing film and store in a storage box at 4°C.

[0155] 10. Capping solution (TdT enzyme mix) reaction

[0156] 1) Take 1.5 ml of DNALobind EP tube and prepare the end-capping reaction solution according to the table below.

[0157] reagents V / ul TdT enzyme 4 10x buffer 40 CoCl2 40 ddTTP 4 Ultrapure water 302 stabilizer 10 Total 400

[0158] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0159] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0160] 4) Add the prepared capping reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the capping reaction program: 37℃, 30min; 4℃, forever.

[0161] 11. Hybridize first-strand sequencing primers and perform first-strand sequencing.

[0162] Prepare the following solution:

[0163] Sequencing reaction solution: 20mM Tris-HCl pH=8.3; 10mM (NH4)2SO4; 50mM KCl; 1mM MnCl2; 100mM glycine; 0.1% Tween; 9°N polymerase; CIP (alkaline phosphatase, bovine intestine); PO or TG fluorescently labeled dN4Ps. The structural formula of PO fluorescently labeled dN4Ps is as follows: The simplified representation of the base is B. The specific structures of PO and TG are shown in Table 1. The composition of the four substrate nucleotides is as follows.

[0164] dA4Ps, containing 100% PO fluorescently labeled nucleotides, have a relative signal intensity (PO band) of 3 after sequencing;

[0165] dG4Ps contain some PO fluorescently labeled nucleotides and some TG fluorescently labeled nucleotides. The ratio of the two types of nucleotides is adjusted so that the relative signal intensity (PO band) after sequencing is 2.

[0166] dC4Ps contain some PO fluorescently labeled nucleotides and some TG fluorescently labeled nucleotides. The ratio of the two types of nucleotides is adjusted so that the relative signal intensity (PO band) after sequencing is 1.

[0167] dT4Ps contain 100% TG fluorescently labeled nucleotides and have a relative signal intensity (PO band) of 0 after sequencing.

[0168] Blocking reaction solution: 20 mM tris(3-hydroxypropyl)phosphine (THPP), 0.5 M NaCl, 50 mM Tris-HCl, pH = 8.6, 0.05% Tween-20;

[0169] Cleaning solution: 20 mM Tris-HCl pH=8.3; 10 mM (NH4)2SO4; 50 mM KCl; 0.1 mM EDTA and 0.1% Tween 20.

[0170] Oil sealant: Novec TM 7500 electronic fluorinated liquid (purchased from 3M) TM ).

[0171] 1) Dilute the first sequencing primer, which is dissolved in 1x TE at a concentration of 100 μM, to 2 μM in an EP tube with hybridization buffer.

[0172] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0173] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles and then clean with 500ul of hybridization solution.

[0174] 4) Add the prepared and mixed sequencing primer solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the sequencing primer hybridization program: 60℃, 7 min; 40℃, 3 min.

[0175] 5) After the reaction is complete, add 500 μL of washing solution to clean the sample. Then perform first-strand sequencing.

[0176] 6) Control the chip temperature to 4℃ and introduce the sequencing reaction solution into the chip;

[0177] 7) Introduce 100-200 μL of sealing fluid into the chip;

[0178] 8) Heat the chip to 55°C and perform nucleotide polymerization to incorporate nucleotides into the 3′ end of the growing nucleic acid chain and terminate further growth; extend for 60 seconds;

[0179] 9) Take a picture, detect the fluorescence signal using a 540nm light source as the excitation wavelength, take a picture, and store the image;

[0180] 10) First, clean off the sealing liquid, add 200μL of deblocking reaction solution into the chip, set the chip temperature to 55℃, and react for 120s to remove the blocking group azidomethyl;

[0181] 11) Cleaning: Introduce 400uL of cleaning solution into the chip and clean it 3 times;

[0182] 12) Repeat steps 6)-11) to perform the next cycle sequencing, for a total of 150 cycles.

[0183] 12. Recombinase polymerase reaction

[0184] 1) Prepare the reaction solution according to the table below.

[0185] reagents V / ul hydration solution 180 Magnesium acetate 15 Ultrapure water 95.25 stabilizer 7.5 Total volume 300

[0186] 2) After preparation, shake to mix well and centrifuge quickly for 2 seconds.

[0187] 3) Clean the chip with 500ul of cleaning solution. If there are bubbles, add 200ul of isopropanol to remove the bubbles before cleaning with 500ul of cleaning solution.

[0188] 4) Add the prepared amplification reaction solution to the chip, place the chip on the PCR instrument, tighten the cap, and select the heating program: 40℃, 60min; 4℃, forever.

[0189] 13. The shearing buffer (FPG enzyme mix) reacts with the second amplification primer.

[0190] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the end-capping reaction solution according to the table below.

[0191] reagents V / ul FPG enzyme 10 10x NEB buffer 40 BSA 4 stabilizer 10 Ultrapure water 336 Total 400

[0192] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0193] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0194] 4) Add the prepared reaction solution to the chip, place the chip on the plate PCR instrument, tighten the cap, and select the shearing reaction program: 37℃, 60min; 4℃, forever.

[0195] 14. Unwinding

[0196] 1) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0197] 2) Add 200 μL of formamide to each chip and react at room temperature (25°C) for 10 min.

[0198] 3) After the reaction is complete, add 1 ml of 1xTE for washing. If sequencing is not performed immediately, seal with sealing film and store in a storage box at 4°C.

[0199] 15. Capping solution (TdT enzyme mix) reaction

[0200] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the end-capping reaction solution according to the table below.

[0201] reagents V / ul TdT enzyme 4 10x buffer 40 CoCl2 40 ddNTP(10mM, each) 8 Ultrapure water 308 Total 400

[0202] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0203] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0204] 4) Add the prepared capping reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the capping reaction program: 37℃, 30min; 4℃, forever.

[0205] 16. Hybridize the second sequencing primers and perform the second-strand sequencing reaction.

[0206] Second-strand sequencing uses the same sequencing primers as first-strand sequencing, but all other required solutions and reaction procedures are the same, so they will not be repeated here.

[0207] 17. Sequencing Results Analysis

[0208] Intensity extraction was performed on the sequencing images collected from the first-strand and second-strand sequencing reactions to obtain the unit signals of the first-strand and second-strand sequencing processes, respectively. The unit signal reflects the sequencing strand generation efficiency; typically, the first-strand unit signal is higher than the second-strand unit signal. In this example, the first-strand unit signal was 403, and the second-strand unit signal was 322. The second-strand unit signal was significantly lower than the first-strand signal, accounting for only 80% of the first-strand unit signal. Consistent with this result, the sequencing accuracy of the second strand was also low, with an AQ30 ratio of only 85.3%.

[0209] Example 3

[0210] Steps 1-10 are the same as in Example 2, and the relevant content of Example 2 will not be repeated here.

[0211] 11. Amplification primer inoculation reaction

[0212] 1) Take 1.5 ml of DNALobind EP tube and prepare the amplification primer inoculation reaction solution according to the table below.

[0213] reagents V / ul Third amplification primer (containing dU cleavage site) 5.5 Click Buffer 285 Total 300

[0214] The 3' end of the third amplification primer contains a spacer (containing 18 ethylene glycol structures) capping, which can prevent primer extension.

[0215] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0216] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0217] 4) Add the prepared amplification primer inoculation reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the click reaction program: 60℃, 30min; 4℃, forever.

[0218] 12. Hybridize the first sequencing primers and perform first-strand sequencing.

[0219] For the specific reaction solution and procedure, please refer to Example 2, which will not be repeated here.

[0220] 13. Third amplification primer decapping reaction

[0221] 1) Take 1.5 ml of DNALobind EP tube and prepare the decapping reaction solution according to the table below.

[0222] reagents V / ul USER enzyme 4 Cutsmart 40 Ultrapure water 346 stabilizer 10 Total 400

[0223] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0224] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0225] 4) Add the prepared shearing reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the shearing reaction program: 37℃, 30min; 4℃, forever.

[0226] 5) The third amplification primer after unsealing contains a 15nt identical sequence to the first amplification primer.

[0227] 14. Primer dephosphorylation reaction

[0228] 1) Take 1.5 ml of DNALobind EP tube and prepare the dephosphorylation reaction solution according to the table below.

[0229] reagents V / ul T4 PNK 4 T4 PNK Buffer 40 Ultrapure water 346 stabilizer 10 Total 400

[0230] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0231] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0232] 4) Add the prepared dephosphorylated reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the reaction program: 37℃, 30min; 4℃, forever.

[0233] 15. Second amplification reaction

[0234] 1) Prepare the reaction solution according to the table below.

[0235] reagents V / ul hydration solution 180 Magnesium acetate 15 Ultrapure water 95.25 stabilizer 7.5 Total volume 300

[0236] 2) After preparation, shake to mix well and centrifuge quickly for 2 seconds.

[0237] 3) Clean the chip with 500ul of cleaning solution. If there are bubbles, add 200ul of isopropanol to remove the bubbles before cleaning with 500ul of cleaning solution.

[0238] 4) Add the prepared amplification reaction solution to the chip, place the chip on the PCR instrument, tighten the cap, and select the heating program: 40℃, 60min; 4℃, forever.

[0239] Steps 16-19 involve shearing, unwinding, end capping, and second-strand sequencing reactions, which are performed in the same manner as steps 13-16 in Example 2, and will not be repeated here.

[0240] 20. Sequencing Results Analysis

[0241] Intensity extraction was performed on the sequencing images collected from the first-strand and second-strand sequencing reactions to obtain the first-strand sequencing unit signal and the second-strand sequencing unit signal, respectively. In this embodiment, the first-strand sequencing unit signal was 397 (basically the same as in Example 2), and the second-strand unit signal was 356, with the second-strand unit signal accounting for 89.7% of the first-strand unit signal, a significant improvement compared to Example 2. Correspondingly, the sequencing accuracy of the second strand was also improved, with the AQ30 ratio increasing to 87.9%. This indicates that providing new 3' end reversibly blocked amplification primers and adjusting the reaction sequence in this embodiment is beneficial for improving the second-strand generation efficiency and the accuracy of second-strand sequencing.

[0242] Example 4

[0243] Steps 1-10 are the same as in Example 2, and the relevant content of Example 2 will not be repeated here.

[0244] 11. Amplification primer inoculation reaction

[0245] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the amplification primer inoculation reaction solution according to the table below, wherein the ratio of the number of the third amplification primer to the number of the fourth amplification primer is 1:1.

[0246]

[0247]

[0248] The 3' ends of the third and fourth amplification primers both contain spacer caps to prevent primer extension.

[0249] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0250] 3) Clean the chip after the end-capping reaction with 500ul of cleaning solution. If there are air bubbles, you can add...

[0251] Use 200ul IPA to remove air bubbles, then use 500ul cleaning solution to clean.

[0252] 4) Add the prepared amplification primer inoculation reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the click reaction program: 60℃, 30min; 4℃, forever.

[0253] 12. Hybridize the first sequencing primers and perform first-strand sequencing.

[0254] For the specific reaction solution and procedure, please refer to Example 2, which will not be repeated here.

[0255] 13. Primer decapping reaction

[0256] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the decapping reaction solution according to the table below.

[0257] reagents V / ul USER enzyme 4 Cutsmart 40 Ultrapure water 346 stabilizer 10 Total 400

[0258] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0259] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0260] 4) Add the prepared shearing reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the shearing reaction program: 37℃, 30min; 4℃, forever.

[0261] 5) The third amplification primer after the unsealed end contains a 15 nt identical sequence to the first amplification primer, and the fourth amplification primer after the unsealed end contains a 15 nt identical sequence to the second amplification primer.

[0262] 14. Primer dephosphorylation reaction

[0263] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the dephosphorylation reaction solution according to the table below.

[0264] reagents V / ul T4 PNK 4 T4 PNK Buffer 40 Ultrapure water 346 stabilizer 10 Total 400

[0265] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0266] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0267] 4) Add the prepared dephosphorylated reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the reaction program: 37℃, 30min; 4℃, forever.

[0268] 15. Second amplification reaction

[0269] 1) Prepare the reaction solution according to the table below.

[0270]

[0271] 2) After preparation, shake to mix well and centrifuge quickly for 2 seconds.

[0272] 3) Clean the chip with 500ul of cleaning solution. If there are bubbles, add 200ul of isopropanol to remove the bubbles before cleaning with 500ul of cleaning solution.

[0273] 4) Add the prepared amplification reaction solution to the chip, place the chip on the PCR instrument, tighten the cap, and select the heating program: 40℃, 60min; 4℃, forever.

[0274] Steps 16-19 involve shearing, unwinding, end capping, and second-strand sequencing reactions, which are performed in the same manner as steps 13-16 in Example 2, and will not be repeated here.

[0275] 20. Sequencing Results Analysis

[0276] Intensity extraction was performed on the sequencing images collected from the first-strand and second-strand sequencing reactions to obtain the first-strand sequencing unit signal and the second-strand sequencing unit signal, respectively. In this embodiment, the first-strand sequencing unit signal was 407 (essentially the same as in Example 3), and the second-strand unit signal was 398, accounting for 98.7% of the first-strand unit signal. Although slightly lower than the first-strand signal, it was very close. Correspondingly, the sequencing accuracy of the second strand was significantly improved, with the AQ30 percentage increasing to 90.1%. This indicates that the provision of two new 3' end reversibly blocking amplification primers in this embodiment, combined with adjustments to the reaction sequence, both contribute to improving the second-strand generation efficiency and the second-strand sequencing accuracy.

[0277] Example 5

[0278] In this embodiment, step 11 is adjusted based on embodiment 4, while steps 1-10 and 12-19 remain unchanged. Specifically:

[0279] 11. Amplification primer inoculation reaction

[0280] 1) Take 1.5 ml of DNA Lobind EP tube and prepare the amplification primer inoculation reaction solution according to the table below, wherein the ratio of the number of the third amplification primer to the number of the fourth amplification primer is 3:1.

[0281] reagents V / ul Third amplification primer (containing dU cleavage site) 15 Fourth amplification primer (containing dU cleavage site) 5 Click Buffer 280 Total 300

[0282] The 3' ends of the third and fourth amplification primers both contain spacer caps to prevent primer extension.

[0283] 2) After preparation, shake to mix well, centrifuge quickly for 2 seconds, and place on ice for later use.

[0284] 3) Clean the reacted chip with 500ul of cleaning solution. If there are bubbles, add 200ul of IPA to remove the bubbles before cleaning with 500ul of cleaning solution.

[0285] 4) Add the prepared amplification primer inoculation reaction solution to the chip, place the chip on a plate PCR instrument, tighten the cap, and select the click reaction program: 60℃, 30min; 4℃, forever.

[0286] 20. Sequencing Results Analysis

[0287] Intensity extraction was performed on the sequencing images collected from the first-strand and second-strand sequencing reactions to obtain the first-strand sequencing unit signal and the second-strand sequencing unit signal, respectively. In this embodiment, the first-strand sequencing unit signal was 402 (basically the same as the value in Example 4), and the second-strand unit signal was 410. The second-strand unit signal was slightly higher than the first-strand signal, accounting for 102.0% of the first-strand unit signal. Correspondingly, the sequencing accuracy of the second strand was also improved, with the AQ30 ratio increasing to 91.3%. This indicates that adjusting the ratio of the number of the two newly provided primers in this embodiment is beneficial to further improve the second-strand generation efficiency and the second-strand sequencing accuracy.

[0288] The above are preferred embodiments of the present invention. Those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments described above. Any obvious improvements, substitutions or modifications made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. A paired-end sequencing method, characterized in that, include: A solid-phase medium is provided, the surface of which contains reaction sites, and the reaction sites are immobilized with a double-stranded nucleic acid molecule containing a target nucleic acid sequence, the double-stranded nucleic acid molecule being obtained by first amplification of the target nucleic acid using amplification primers; the amplification primers contain cleavage sites. The double-stranded nucleic acid molecule is subjected to a first treatment to obtain a first strand that is essentially single-stranded for sequencing; new amplification primers are provided to the solid-phase medium, and the 3' ends of the new amplification primers are blocked. A sequencing reaction solution is provided for first-strand sequencing. The sequencing reaction solution contains a substrate nucleotide, which is a nucleotide with a 5' end phosphorylated fluorescently switched label and a 3' end containing a reversible termination group. Unblock the new amplification primers and perform a second amplification to obtain double-stranded nucleic acid. The nucleic acid double strands are subjected to a second processing to obtain a second strand that is essentially single-stranded for sequencing. A sequencing reaction solution is provided for second-strand sequencing. The sequencing reaction solution contains a substrate nucleotide, which is a nucleotide with a 5' end phosphorylated fluorescently switched label and a 3' end containing a reversible termination group. The paired-end sequencing of the target nucleic acid was completed by determining the sequences of the first and second strands.

2. The method according to claim 1, characterized in that, The structure of the substrate nucleotide is shown in formula (Ⅰ): Wherein, Y is O or S, B is a heterocyclic base, and n is an integer from 0 to 6; R is selected from azidomethyl, amino, allyl, substituted dithioalkyl, substituted methoxymethyl, o-nitrobenzyl, coumarin, phosphate nitrile ethyl ester, trimethylsilyl, tetrahydropyranyl, azido, alkyl hydroxyamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, or vinyl; Fluorogenic Dye is selected from anthracene, phenoxazine, acridine, or coumarin with fluorescence switching properties, wherein the anthracene with fluorescence switching properties includes one or more of oxanthracene-fluorescein, carbamate-Beijing orange, silanthracene, germananthracene, phosphoxanthracene, or thioanthracene.

3. The method according to claim 1, characterized in that, The first amplification includes a first amplification primer and a second amplification primer. The first treatment is applied to the first amplification primer. The new amplification primer includes a third amplification primer. The unblocked third amplification primer contains at least a portion of the consistent sequence of the first amplification primer. The length of the consistent sequence is at least 15 nt, preferably at least 20 nt.

4. The method according to claim 1, characterized in that, The first amplification includes a first amplification primer and a second amplification primer. The first treatment is applied to the first amplification primer. The new amplification primer includes a third amplification primer and a fourth amplification primer. The unblocked third amplification primer contains at least a partially consistent sequence of the first amplification primer, and the unblocked fourth amplification primer contains at least a partially consistent sequence of the second amplification primer. The length of the consistent sequence is at least 15 nt, preferably at least 20 nt.

5. The method according to claim 4, characterized in that, The ratio of the number of the third amplification primer to the number of the fourth amplification primer is 1:1 to 5:1, preferably 2:1 to 3:

1.

6. The method according to claim 1, characterized in that, The 3' end of the new amplification primer is closed by fixing the new amplification primer to the surface of a solid medium and then modifying the 3' end of the primer, including phosphorylation modification or ddNTP modification.

7. The method according to claim 1, characterized in that, The 3' end of the new amplification primer is closed, meaning that the 3' end of the new amplification primer has been modified before it is fixed to the surface of the solid medium. The modification is selected from one or more of phosphorylation modification, ddNTP modification, inter-arm modification, amino modification, or biotin modification.

8. The method according to claim 6 or 7, characterized in that, The new amplification primer contains a cleavage site, which is used to unblock the new amplification primer. The cleavage site is different from the cleavage site used in the second treatment.

9. The method according to claim 1, characterized in that, The reaction sites comprise multiple reaction volumes created by multiple reaction chambers disposed in a solid medium, wherein the double-stranded nucleic acid molecule containing the target nucleic acid sequence is immobilized in the reaction volume; after the sequencing reaction solution is delivered to each reaction volume, each reaction volume may be sealed and / or separated from other reaction volumes on the array; The emission signal from the fluorescent switching label can then be detected and / or recorded for each reaction volume.

10. The method according to any one of claims 1-9, characterized in that, The method further includes removing the reversible termination group at the 3' end of the incorporated nucleotide before the next sequencing cycle, so that the nucleotide becomes an extendable state.

Images