CN114438185B

CN114438185B - Method for double-end amplification sequencing of chip surface

Info

Publication number: CN114438185B
Application number: CN202210103676.8A
Authority: CN
Inventors: 乔朔; 辛宇; 石磊; 康力; 王晓洁
Original assignee: Peking University
Current assignee: Peking University
Filing date: 2022-01-28
Publication date: 2024-05-31
Anticipated expiration: 2042-01-28

Abstract

The invention relates to a method for double-end amplification sequencing on the surface of a chip, which is characterized in that after the sequencing of a first chain is finished, amplified oligonucleotides are provided on the surface of the chip again for generating a second chain, the number of the generated second chain can be controlled by controlling the number of the provided amplified oligonucleotides, and the sequencing signal of the second chain is increased, so that the sequencing accuracy is improved; the method of the invention can also be compatible with other high-throughput sequencing technologies, and has stronger compatibility.

Description

Method for double-end amplification sequencing of chip surface

Technical Field

The invention relates to a method for double-end amplification sequencing of a chip surface, and belongs to the field of gene sequencing.

Background

The high-throughput sequencing technology at the present stage has rapid development, becomes a common analysis method for life research at present, has the characteristics of high throughput, high detection speed, flexibility, multiple purposes and low cost, and the technology needs to exponentially amplify target DNA (deoxyribonucleic acid) through amplification so as to realize the aim of simultaneously detecting hundreds of thousands to millions of DNA molecules, so that one sequencing technology corresponds to one amplification mode. At present, single-ended sequencing is simpler in the field of high-throughput sequencing, namely, only one sequencing primer is needed, extension sequencing is carried out from the direction of 5'-3' of the primer during sequencing, signals can be read only in one direction, amplification is simpler, and a large number of samples to be tested can be obtained by amplifying target DNA once through PCR or other amplification technologies. But the quality of single-ended sequencing decreases as sequencing proceeds, the more inaccurate the sequencing sequence is, and the longer the read is limited.

The double-End sequencing technology refers to adding sequencing primer binding sites on joints at two ends when constructing a DNA library to be tested, and after the first strand sequencing is completed, a reading and sequencing Module (Paired-End Module) is used for guiding the regeneration and amplification of the second strand so as to reach the template quantity used for the second round of sequencing, and the second strand sequencing is performed. When double-end sequencing is carried out on the same nucleic acid molecule, for example, a common double-end sequencing method of Illumina (see patent CN 101663405B), the surface of a solid phase medium is connected with two amplification primers, each amplification primer contains different shearing sites, and double-stranded DNA formed by amplification is respectively linearized by shearing the shearing sites and then sequenced; after the first strand sequencing is finished, the sheared amplification primer is remained on the surface of the medium, and the second strand generation reaction is carried out by using the amplification primer for the two-strand sequencing. This conventional technical route has obvious disadvantages: in the process of sequencing the first strand, through tens of rounds of sequencing reaction cycles and even hundreds of rounds of sequencing reaction cycles, the sheared amplification primers remained on the surface of the medium inevitably lose, are combined with proteins such as polymerase and the like in a non-specific way and are not blocked successfully to be extended in a non-specific way, and the factors greatly reduce the amplification primers which can be used for generating the second strand, seriously influence the generation efficiency of the second strand, reduce the sequencing signals of the second strand and finally reduce the sequencing accuracy.

In order to solve the problems, the invention provides a new technical idea, namely, the amplification primer is thoroughly sheared off as close to the 5' end of the amplification primer as possible in the shearing reaction before the first strand sequencing, and after the first strand sequencing is finished, the new amplification primer is provided again for the generation of the second strand, so that the generation amount of the second strand is increased, and the sequencing signal and the sequencing accuracy are improved.

Disclosure of Invention

The invention discloses a method for double-end amplification sequencing of a chip surface, which is characterized by comprising the following steps of:

(1) Providing at least two amplification oligonucleotides immobilized on the surface of the support medium, i.e., providing at least a first amplification oligonucleotide comprising one cleavage site and a second amplification oligonucleotide comprising one cleavage site, the two cleavage sites being different from each other; the surface of the supporting medium is modified with chemical groups;

(2) Providing a single stranded template polynucleotide, hybridizing the single stranded template polynucleotide to a first amplification oligonucleotide on the surface of a support medium; the two ends of the single-stranded template polynucleotide contain public linker sequences, namely a linker sequence 1 and a linker sequence 2, wherein at least part of the sequences of the linker sequence 1 and the first amplification oligonucleotide are complementarily paired; the adaptor sequence 2 and the second amplification oligonucleotide are identical in at least part of the sequence; initially extending the first amplification oligonucleotide to generate an extension product complementary to the template polynucleotide; unwinding to obtain a polynucleotide single chain with the medium surface carrying complementary pairing with the template polynucleotide;

(3) Amplification: providing an amplification reactant, performing medium surface amplification, and generating a plurality of double-stranded polynucleotides immobilized on the medium surface, wherein the double-stranded polynucleotides comprise a first strand and a second strand;

(4) Shearing and end capping: interrupting the first amplification oligonucleotide at a cleavage site position by acting on the first amplification oligonucleotide, selectively removing a second strand in the double-stranded polynucleotide, and generating an extendable 3' -end; adding a blocking reagent to block the 3' end of the nucleic acid chain or the oligonucleotide on the surface of the medium;

(5) First strand sequencing: hybridizing a first sequencing primer and sequencing; unwinding;

(6) Re-supplying an amplification oligonucleotide to the chip surface for the generation of a second strand, the amplification oligonucleotide comprising at least the first amplification oligonucleotide or a first amplification oligonucleotide that does not comprise a cleavage site;

(7) Generating a second strand complementary to the first strand immobilized on the surface of the medium;

(8) Shearing and end capping: interrupting the second amplification oligonucleotide at a cleavage site position by acting on the second amplification oligonucleotide, selectively removing the first strand of the double-stranded polynucleotide, and generating an extendable 3' -end; adding a blocking reagent to block the 3' end of the nucleic acid chain or the oligonucleotide on the surface of the medium;

(9) Second strand sequencing: hybridizing a second sequencing primer and sequencing.

According to a preferred embodiment, the surface of the supporting medium is provided with a concave structure which is distributed discretely, and is a micro-reaction chamber for reaction, and the shape of the micro-reaction chamber is cylindrical, truncated cone-shaped, groove, truncated cone-shaped, hexagonal column-shaped or a combination of the micro-reaction chamber and the groove.

According to a preferred embodiment, the support medium is an inert substrate or matrix whose material includes, but is not limited to, glass, silicon, silica, optical fibers or bundles of optical fibers, resins, ceramics, metals, nitrocellulose, polyethylene, polystyrene, copolymers of styrene with other materials, polypropylene, acrylic, polybutylene, polyurethane.

According to a preferred embodiment, the support medium comprises an inert substrate or matrix and a mediator material directly attached to the amplification oligonucleotide and linked to the inert substrate or matrix by covalent or non-covalent forces; the medium materials include, but are not limited to, hydrogel layers, hydrogel microspheres, and magnetic microspheres; materials for the inert substrate or matrix include, but are not limited to, glass, silicon, silica, optical fibers or bundles of optical fibers, resins, ceramics, metals, nitrocellulose, polyethylene, polystyrene, copolymers of styrene with other materials, polypropylene, acrylic, polybutylene, polyurethane.

According to a preferred embodiment, said chemical groups supporting surface modification of the medium include amino, carboxyl, epoxy, hydroxyl, aldehyde, azide, alkyne, cycloalkyne, maleimide, succinimide, mercapto, etc.; the amplification oligonucleotide is immobilized on the surface of the support medium by reaction with the chemical group.

According to a preferred embodiment, the cleavage site allows enzymatic cleavage, chemical cleavage or photochemical cleavage.

According to a preferred embodiment, wherein the cleavage site comprises a site cleaved with a nicking endonuclease.

According to a preferred embodiment, the shearing comprises contacting the support medium surface with a composition comprising at least one enzyme to create an abasic site at the shearing site, wherein the shearing occurs at the shearing site.

According to a preferred embodiment, the amplification oligonucleotide comprises a uracil base or an 8-oxoguanine base or a deoxyhypoxanthine base or a tetrahydrofuran modified base.

According to a preferred embodiment, wherein the at least one enzyme that produces an abasic site at the cleavage site comprises uracil DNA glycosylase and an endonuclease or endonuclease iv selected from the group consisting of DNA glycosylase-lyase endonucleases viii or Fpg glycosylase.

According to a preferred embodiment, the cleavage site is selected from uracil bases, 8-oxoguanine bases, deoxyinosine bases, tetrahydrofuran modified bases, ortho-dihydroxyl modified phosphoramidite sites, disulfide groups, azo groups, azido groups, peptide bonds, one or more ribonucleotides, ketals, acetals, diphenylsiloxanes, carbonates, carbamates, and the like.

According to a preferred embodiment, after completion of the cleavage reaction described in step (4) and step (8), it is desirable to generate an extendable 3' end, i.e.: if a phosphate group is formed at the 3 'end after the cleavage reaction, it is necessary to treat the 3' end phosphate group formed after cleavage with a phosphokinase or phosphatase enzyme, including T4 polynucleotide kinase, to cleave the 3 'end phosphate group and generate an extendable 3' end.

According to a preferred embodiment, the amplification is one of loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), recombinase-mediated isothermal nucleic acid amplification (RAA), nicking endonuclease isothermal amplification (NEAR), rolling Circle Amplification (RCA), nucleic acid sequence dependent amplification (NASBA), helicase Dependent Amplification (HDA), strand Displacement Amplification (SDA), bridge amplification or PCR.

According to a preferred embodiment, the amplification reagents comprise a polymerase and dNTPs.

According to a preferred embodiment, the amplification reagents comprise a recombinase and a single-stranded binding protein.

According to a preferred embodiment, the 5' end of the re-supplied amplification oligonucleotide of step (6) is modified with a specific group, immobilized on the surface of the support medium by reaction with a chemical group on the surface of the support medium, resulting in a complete amplification oligonucleotide which is hybridized to the first strand; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto and the like.

According to a preferred embodiment, the re-provided amplification oligonucleotides comprise a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotides to the number of second amplification oligonucleotides being any number between 1 and 10, preferably any number between 1 and 5; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site.

According to a preferred embodiment, step (7) comprises providing an amplification reaction, performing amplification or extension, generating a second strand complementary to the first strand immobilized on the surface of the medium.

According to a preferred embodiment, the re-provided amplification oligonucleotide of step (6) comprises a first amplification oligonucleotide in the liquid phase, which can hybridize to the first strand, the first amplification oligonucleotide being modified at the 5' end with a specific group; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto and the like.

According to a preferred embodiment, step (7) comprises hybridizing the liquid phase of the first amplification oligonucleotide to the first strand to provide an amplification reactant, extending a second strand; the second strand is immobilized on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium.

According to a preferred embodiment, step (7) comprises hybridizing the liquid phase of the first amplification oligonucleotide to the first strand, immobilizing the first amplification oligonucleotide on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium; providing an amplification reaction, performing amplification or extension of the surface of the medium, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

According to a preferred embodiment, step (7) comprises providing a liquid phase amplification oligonucleotide modified at the 5' end with a specific group, immobilizing the liquid phase amplification oligonucleotide and the first amplification oligonucleotide hybridized to the first strand in step (6) on the surface of the support medium by reaction with a chemical group on the surface of the support medium; providing an amplification reactant, and performing medium surface amplification or extension to generate a second chain fixed on the surface of the medium; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto and the like.

According to a preferred embodiment, the liquid phase amplification oligonucleotide modified at the 5' end with a specific group provided in step (7) comprises at least a first amplification oligonucleotide and may further comprise a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotide to the number of second amplification oligonucleotide being any number between 1 and 10, preferably any number between 1 and 5; the re-provided first amplification oligonucleotide comprises or does not comprise a cleavage site and the re-provided second amplification oligonucleotide comprises a cleavage site thereon.

According to a preferred embodiment, the template polynucleotide comprises a first index and a second index, the method further comprising sequencing the first index and the second index.

According to a preferred embodiment, characterized in that the sequencing is sequencing-by-synthesis or sequencing-by-ligation, preferably fluorescence sequencing.

The invention provides a gene sequencing method, which is characterized in that nucleic acid molecules to be detected are fragmented, a library is constructed to obtain template polynucleotides, and the amplification sequencing is performed according to the method described in any one of the previous claims.

The invention has the advantages that

Compared with the prior art, the double-end amplification sequencing method disclosed by the invention has the following advantages:

1. The method of the invention realizes the regeneration of the second strand by providing the amplification oligonucleotide again to the surface of the chip after the sequencing of the first strand is completed, avoids the use of the amplification oligonucleotide which is worn and generates nonspecific binding or nonspecific extension in the prior art to regenerate the second strand, and can control the generation quantity of the second strand and increase the sequencing signal of the second strand by controlling the quantity of the provided amplification oligonucleotide, thereby improving the sequencing accuracy.

2. The method of the invention can be compatible with other high-flux sequencing technologies, and has stronger compatibility.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described. It is apparent that the drawings in the following description are only some embodiments consistent with the present invention, and it is possible for those of ordinary skill in the art to obtain drawings corresponding to other embodiments from these drawings without inventive effort.

FIG. 1 shows a schematic flow chart of double-end amplification sequencing on the surface of a chip, wherein 101 represents a first amplification oligonucleotide immobilized on the surface of a medium, 102 represents a second amplification oligonucleotide, one end of a primer with an arrow represents the 3' end of the primer, 103 represents a chemical group for modifying the surface of the medium, a black dot represented by 104 represents one cleavage site on the first amplification oligonucleotide, a gray dot represented by 105 represents one cleavage site on the second amplification oligonucleotide, and the two cleavage sites are different from each other.

FIGS. 2A-B are graphs showing the brightness of microspheres and the efficiency of second strand formation when fluorescent-labeled probes were hybridized after first strand and second strand formation in example 1. FIG. 2A is a left graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the first strand is generated, and FIG. 2A is a right graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the second strand is generated; fig. 2B is a second chain generation efficiency graph plotted by MATLAB.

FIGS. 3A-B are graphs showing the brightness of microspheres and the efficiency of second strand formation when fluorescent-labeled probes were hybridized after first strand and second strand formation in example 2. FIG. 3A is a left graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the first strand is generated, and FIG. 3A is a right graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the second strand is generated; fig. 3B is a second chain generation efficiency plot of MATLAB.

FIGS. 4A-B are graphs showing the brightness of microspheres and the efficiency of second strand formation when fluorescent-labeled probes were hybridized after first strand and second strand formation in example 3. FIG. 4A is a left graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the first strand is generated, and FIG. 4A is a right graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the second strand is generated; fig. 4B is a second chain generation efficiency plot of MATLAB.

FIGS. 5A-B are graphs showing the brightness of microspheres and the efficiency of second strand formation when fluorescent-labeled probes were hybridized after first strand and second strand formation in example 4. FIG. 5A is a left graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the first strand is generated, and FIG. 5A is a right graph showing the brightness of the microsphere when the fluorescent label probe is hybridized after the second strand is generated; fig. 5B is a second chain generation efficiency plot of MATLAB.

Detailed Description

The invention is described in further detail below with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof. The size and shape of the various parts in the figures are not represented to true scale and are only intended to illustrate the present invention.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" is not limiting and should be interpreted as having an open meaning, that is, it should be interpreted synonymously with the phrase "at least including".

1. Definitions and terms

In the present invention, the term "support medium" refers to any insoluble inert substrate or matrix to which nucleic acid molecules may be attached, such as glass, silicon (e.g., silicon wafers), ceramics, resins, silica-based materials, plastics, nylon, nitrocellulose, metals, optical fibers or bundles of optical fibers, polyethylene, polystyrene, copolymers of styrene and other materials, polypropylene, acrylic, polybutylene, polyurethane, latex beads, dextran beads, porous plates, and the like. The shape of the surface is optional and includes, for example, porous, planar, or spherical as appropriate for the particular application. Preferably, a support medium is mounted inside the flow cell to allow its interaction with the plurality of reagent solutions.

In some preferred embodiments, the "support medium" includes the inert substrate or matrix described above, to which the nucleic acid molecule is attached via a mediator material having a particular chemical group modification, to which the nucleic acid molecule may be covalently attached, and to which the mediator material may be immobilized via covalent or non-covalent forces, including, but not limited to, for example, hydrogel layers, hydrogel microspheres, and the like, to which the inert substrate may be glass. In particular, the medium material may also comprise magnetic microspheres, which are confined to the inert substrate or matrix surface by external forces.

The micro reaction chamber refers to a microstructure of a common sequencing chip, and is also called a micro pit, a micro well and the like. Generally, the concave structures are in discrete distribution, and the shape is a cylinder, a truncated cone, a groove, a truncated cone-like structure, a hexagonal column-like structure or a combination thereof. Each micro-chamber is a reaction chamber that corresponds to a data point when fed back to the sequencing data. This is a common fact in sequencing reactions.

In the present invention, "amplification" refers to amplification in the sense of the art. Amplification refers to gene amplification, which, by a certain technique, increases the copy number of a gene. The term "template polynucleotide" as used herein includes nucleic acid fragments to be detected, and may also be referred to as amplification templates. The amplified template is a fragment to be amplified. The sequence of the template may be unknown. From the above statement, it is known that the amplification process can be performed by interrupting the DNA of a long fragment at the time of sequencing, forming a small fragment, and then ligating the adaptor sequence. Fragmentation of the test molecule may be performed using any of a variety of techniques known in the art, for example, sonication, nebulization, physical shearing, chemical cleavage, or enzymatic hydrolysis, among others. Throughout the process, there is no limitation to the DNA fragment or nucleic acid sequence fragment to be detected, and any sequence may be used for this operation. This is also a concept of complete sequencing.

In the present invention, the "hybridization" is a conventional procedure of molecular biology, i.e., base pairing of two single-stranded DNA or RNA, specifically, for example, hybridization of a template polynucleotide single strand with an amplification primer on the surface of a medium to change from a liquid phase to a solid phase.

In the present invention, the term "isothermal amplification" or "isothermal amplification" is a novel in vitro nucleic acid amplification technique developed subsequent to PCR techniques. Compared with the traditional PCR technology, isothermal amplification has the remarkable advantages that: the detection line is low, temperature change is not needed, the amplification speed is high, the reaction temperature is generally low, and the requirement on high temperature resistance of the chip is reduced.

In the present invention, a "primer" is often used in an amplification reaction. The present invention relates to at least two amplification primers. The 5' end of the amplification primer is immobilized on the surface of the support medium, i.e., the solid phase primer. The two primers function not only to pair with template binding, but also to attach the other end of the DNA strand to the surface of the medium in the presence of a recombinase. Thus, during the amplification process, a process of amplifying the surface of the medium is formed. There is no special requirement for the design and synthesis of the primers. The requirements of different amplification reactions for the number of primers etc. are different. The amplification reaction involved in the present invention may use at least two forms of primers. The specific design of the primers may vary.

The term "amplification oligonucleotide" refers to a sequence, including amplification primers, that may be composed entirely of natural nucleotides or modified nucleotides (e.g., 8-oxoguanine, methylated nucleotides, etc.), and may also include necessary non-nucleoside acid spacers, including, but not limited to, disulfide-or dihydroxy-or azo-or azide-group-containing units, and the like.

In the present invention, the term "polynucleotide" may be used equally to "nucleic acid", and the number of nucleotides constituting "polynucleotide" is large as compared to "oligonucleotide", which usually consists of less than 50 nucleotides.

In the present invention, the term "polymerase" is substantially consistent with its conventional meaning in the art and also includes, for example, enzymes that use nucleic acids as template strands to produce replication of complementary nucleic acid molecules. Typically, a DNA polymerase binds to a template strand, moves along the template strand, and sequentially adds nucleotides to the free hydroxyl groups at the 3' end of the nascent strand of the nucleic acid. RNA polymerase functions mainly in the transcription process, and usually synthesizes RNA molecules using DNA as a template. The polymerase may use primers to initiate chain growth. In addition, some polymerases have strand displacement functions, with the activity of removing complementary strands from the template strand read by the polymerase. Exemplary strand displacement polymerases include, but are not limited to, bst DNA polymerase (large fragment), phi29 DNA polymerase, sau DNA polymerase, and the like, and some useful polymerases have been modified, by mutation, or the like, to reduce or eliminate 3 'and/or 5' exonuclease activity.

In the present invention, the term "recombinase" is consistent with its conventional meaning in the art, in that the recombinase is assembled onto ssDNA (e.g., primers) to form a helical fiber. Exemplary recombinases include, for example, recA protein, T4 uvsX recombinase, and homologous proteins or protein complexes such as SC-recA, BS-recA, rad51, or functional variants thereof.

In the present invention, a "first strand" is a single-stranded polynucleotide that is sequenced in a first round of sequencing reactions; the "second strand" is the single stranded polynucleotide that is sequenced in the second round of the sequencing reaction. The first strand and the second strand are substantially complementarily paired to the same double-stranded nucleic acid molecule.

In the present invention, the term "cleavage site" is used in a broad sense, and any cleavage method is used to cleave a double-stranded nucleic acid molecule at such a site, so that only one polynucleotide strand remains on the surface of the support medium, and such a site may be referred to as a "cleavage site". The shearing method includes, but is not limited to: photochemical cleavage, suitable chemical cleavage, suitable enzymatic cleavage, cleavage of ribonucleotides, cleavage of abasic sites, enzymatic digestion with nicking endonucleases, cleavage of hemimethylated DNA, and the like.

In the present invention, "light shearing" includes any method of shearing a single strand in a nucleic acid to be tested by using light energy. The aforementioned cleavage sites may be located in non-nucleotide chemical spacer units in the nucleic acid to be tested. The chemical spacer units include PC spacer phosphoramidite (4- (4, 4' -dimethoxytrityl) butyrylaminomethyl) -1- (2-nitrophenyl) -ethyl ] -2-cyanoethyl- (N, N-diisopropyl) -phosphoramidite available from GLEN RESEARCH company (Sterling, va., USA) which can be cleaved by exposure to an ultraviolet light source.

This spacer unit can be attached to the 5' end of the single strand of nucleotides, along with phosphorothioate groups that allow attachment to a solid surface, using standard techniques for chemical synthesis of oligonucleotides.

In the present invention, "chemical cleavage" includes any method that utilizes chemical reactions (including, but not limited to, redox reactions, hydrolysis reactions, enzymatic reactions, etc.) to facilitate/effect cleavage of single-stranded polynucleotides. In general, single-stranded polynucleotides may include one or more non-nucleotide chemical moieties and/or non-natural nucleotides and/or non-natural backbone linkages to allow for the performance of a chemical cleavage reaction.

Typically, the chemical cleavage site may be a disulfide group, and the disulfide bond may be cleaved using a chemical reducing agent, such as tris (2-carboxyethyl) phosphonium hydrochloride (TCEP), mercaptoethanol, DTT, cysteine, and the like. Typically, the chemical cleavage site may be a peptide bond, and the cleavage reaction is completed by an enzymatic reaction using enzymes that promote hydrolysis of the peptide bond, including proteinase K and the like.

Typically, the chemical cleavage site may be an oxidative chemical linking group, including a diol linking unit, the number of which may be one or more; any material that promotes glycol cleavage may be used for shearing, preferably periodate (e.g., aqueous sodium periodate) or potassium permanganate; after treatment with a cleavage agent (e.g., periodate) to cleave the diol, the cleavage product may be treated with a "capping agent" to neutralize reactive species generated in the cleavage reaction. Suitable capping agents for this purpose include, but are not limited to, ethanolamine, triethylamine, triethanolamine, arginine, lysine, cysteamine, and the like. In a preferred embodiment, a capping agent (e.g., ethanolamine) may be mixed with a cleavage agent (e.g., periodate) such that the reactive species is capped once formed. One or more diol linking units can be incorporated into the amplification oligonucleotide using standard methods for automated chemical DNA synthesis. Typically, the chemical cleavage site may be a reductive cleavage type linker, including but not limited to a unit containing an azo or azide group. For the chemical connecting unit containing azo groups, the shearing reaction can be completed by utilizing sodium dithionite solution treatment, the sheared residual end is inert, the end capping reaction is not needed, and the method is very convenient; for chemical linking units containing azide groups, the cleavage reaction can be accomplished by treatment with TCEP or hydrazine. Standard methods for automated chemical DNA synthesis can be used to incorporate units containing azo groups or azide groups into amplification oligonucleotides.

Typically, the chemical cleavage site may also be an acid-sensitive linking group, including but not limited to ketal, acetal, diphenylsiloxane, carbonate, carbamate, and the like. Any reaction substance or reaction system that promotes hydrolysis of ketals, acetals, carbonates, carbamates or diphenylsiloxanes may be used for shearing, preferably under reaction conditions of from 5 to 30 minutes at room temperature or 37 ℃ in an acidic buffer system having a pH of from 2 to 3, to effect efficient cleavage. The main product after cutting is a connecting unit with hydroxyl (or amino, for carbamates) at the tail end, and the byproducts are inert substances such as acetone (or other ketone substances), diphenyl silanol, carbon dioxide and the like, so that the byproducts cannot participate in subsequent DNA synthesis reaction, and are removed without other steps.

As used herein, an "abasic site" refers to a location in a nucleic acid molecule from which a base component has been removed. Abasic sites may naturally occur in DNA under physiological conditions by hydrolysis of nucleoside residues, and may also be formed chemically under artificial conditions (e.g., by the action of enzymes). Once the abasic site is formed, the abasic site can be cleaved (e.g., by exposure to heat or alkali treatment with an endonuclease or other single-stranded cleaving enzyme) using a suitable cleavage method, thereby providing a means for site-specific cleavage amplification of the oligonucleotide. One of ordinary skill in the art will recognize that the use of heat or alkali may potentially denature nucleic acid molecules and, thus, may not be a preferred embodiment.

In a preferred embodiment, abasic sites can be generated at predetermined positions of the amplification oligonucleotide by first incorporating deoxyuridine (U) at predetermined cleavage sites for cleavage, and uracil bases can then be removed using Uracil DNA Glycosylase (UDG) to generate abasic sites at specific positions. The strand comprising the abasic site can then be cleaved at the abasic site by treatment with an endonuclease (e.g., endo IV endonuclease, AP lyase, FPG glycosylase/AP lyase, endo VIII glycosylase/AP lyase), heat or alkali.

In addition to deoxyuridine, abasic sites can be created on unnatural/modified deoxyribonucleotides and sheared in a similar manner by treatment with endonucleases, heat or bases. For example, 8-oxoguanine can be converted to an abasic site by exposure to FPG glycosylase; deoxyinosine can be converted to an abasic site by exposure to AlkA glycosylase; the resulting abasic sites can then be cleaved, typically by treatment with a suitable endonuclease (e.g., endo IV, AP lyase). It should be noted that the non-natural/modified nucleotides in the present invention should suffice in a polymerase replication reaction that can be used in an amplification reaction, since they will be incorporated into an amplification oligonucleotide for amplification purposes.

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 a specific embodiment, the USER reagent available from NEW ENGLAND Biolabs is used to form a single nucleotide gap at the uracil base in the amplified oligonucleotide, and treatment with an endonuclease generates a 3 '-phosphate moiety at the cleavage site, which 3' -phosphate can be removed by a suitable phosphatase (e.g., alkaline phosphatase) if desired.

In the field of molecular biology, the use of 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 the field of molecular biology. The method can use essentially any nicking endonuclease, as required to contain the appropriate recognition sequence in the cleavage site present on the amplification oligonucleotide.

One or more ribonucleotides are incorporated into the amplification oligonucleotide, and phosphodiester bonds between deoxyribonucleotides and ribonucleotides are selectively cleaved by suitable cleavage reagents, including ribonucleases, to effect cleavage of the nucleic acid molecule to be detected at a specific locus into a single-stranded sequencing template. For example, the single strand of nucleotides to be cleaved comprises a ribonucleotide to provide a useful cleavage site. Suitable shearing agents include, but are not limited to: metal ions, such as rare earth ions, are also effective, particularly la3+, tm3+, yb3+, lu3+ ions, either Fe (3) or Cu (3) or exposed to elevated pH, for example, treated with a base such as sodium hydroxide. It is particularly noted that the suitable cleavage reagent is not capable of cleaving the phosphodiester bond between two deoxyribonucleotides under the same conditions.

The basic composition of ribonucleotides is generally not important, but can be chosen to optimize chemical (or enzymatic) cleavage. For example, if cleavage is to be performed by exposure to metal ions, particularly rare earth metal ions, rUMP or rCMP is generally preferred. For cleavage with ribonucleases, it is preferable to include two or more consecutive ribonucleotides, for example, 2 to 10 or 5 to 10 consecutive ribonucleotides. The exact sequence of ribonucleotides is generally not critical, and suitable RNases include, for example, RNase A, which cleaves after C and U residues. Thus, when cleaved with RNase A, the cleavage site must comprise at least one C or U ribonucleotide. Amplification oligonucleotides incorporating one or more ribonucleotides can be readily synthesized with suitable ribonucleotide precursors using standard techniques for oligonucleotide chemical synthesis.

In the present invention, the term "hemimethylated DNA" refers to an oligonucleotide comprising one or more methylated nucleotides that are opposite to unmethylated deoxyribonucleotides on the complementary strand, such that annealing of the two strands results in a hemimethylated duplex structure. For cleavage of the nucleic acid, site-specific cleavage can be achieved by cleavage with an endonuclease; the endonuclease is specific for a recognition sequence comprising methylated nucleotides. Amplification oligonucleotides incorporating one or more methylated nucleotides can be prepared using appropriate methylated nucleotide precursors using standard techniques of automated DNA synthesis.

As used herein, "capping" is a common step in solid phase amplification, and is the step of transferring a nucleoside bearing a non-extendable 3 'group to the 3' end of a solid phase nucleic acid (e.g., an unextended amplified oligonucleotide) using a tool enzyme such as terminal transferase (TdT), which terminal does not continue to react, thereby reducing the occurrence of side reactions. Terminal transferase (TdT) is a template independent DNA polymerase that catalyzes the binding of deoxynucleotides to the 3' hydroxyl end of a DNA molecule. Single and double stranded DNA molecules with protruding, recessed or smooth ends can be used as substrates for TdT, and the tailing length can reach 5-300nt. The capping reaction is performed prior to the sequencing reaction.

2. Summary of the invention

In the method of the invention, firstly, an amplifying oligonucleotide is planted on the surface of a supporting medium, and the amplifying oligonucleotide can be fixed on the surface of the supporting medium through reaction with chemical groups modified on the surface of the supporting medium, wherein the chemical groups comprise: amino, carboxyl, epoxy, hydroxyl, aldehyde, azide, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto, etc.

According to a preferred embodiment, when the mediator material is a microsphere, the diameter of the microsphere should be matched to the size of the microreactor chamber, i.e. the microsphere diameter should be smaller than the size of the microreactor chamber and not smaller than half the size, so that the microsphere can enter the microreactor chamber, and more than one microsphere cannot enter one microreactor chamber, i.e. the size of the microreactor chamber is larger than the diameter of the sequencing microsphere but smaller than 2 times the diameter of the microsphere.

According to a preferred embodiment, the microspheres have a size of 0.2-5 microns, preferably 0.3-3 microns, more preferably 0.35-2.5 microns.

In some preferred embodiments, the microsphere which is immobilized with at least two amplification oligonucleotides and contains modification of specific chemical groups can be loaded on the surface of the chip which is processed into the micro-reaction chamber and modified, so that most of the micro-reaction chambers of the chip are provided with the microsphere, and the outside of the micro-reaction chamber is not provided with the microsphere. Preferably, at most one microsphere is loaded in the microreactor chamber.

In the initial stage of amplification, it is necessary to immobilize the amplified template polynucleotide to the surface of the medium, specifically by means of a common linker sequence at both ends of the template (i.e. linker sequence 1 and linker sequence 2), which is at least partially complementary to the amplified oligonucleotide on the surface of the medium and thus can hybridize to the surface of the support medium according to the principle of base complementary pairing. Prior to hybridization, the double-stranded library is unwound to single strands (if single-stranded, it can be hybridized directly) and then complementarily paired with the solid phase primer. After hybridization, excess template is washed off and enters into a reaction solution containing DNA polymerase for reaction, so that the solid phase amplification primer extends out of a complete complementary pair of template strands according to the hybridized template polynucleotide. Next, a helicase is added to react and the unwound polynucleotide strand is washed away. Thus, the whole chip has no free DNA in liquid phase, and all template DNA is copied to the surface of the support medium.

In the present invention, the adaptor sequence 1 and the first amplification oligonucleotide are complementary to each other in at least a part of the sequences. This allows hybridization in the form of complementary pairs when the amplified templates hybridize. The adaptor sequence 2 and at least part of the sequence of the second amplification oligonucleotide are coincident. After hybridization, the reverse DNA strand of the amplified template, or the complementary strand pair, is obtained by unwinding, and the adaptor sequence is also reverse or complementary paired. Thus, the reverse adaptor sequence is required to bind to the second amplification oligonucleotide during the subsequent amplification reaction. That is, at least part of the sequences of the adaptor sequence 2 and the second amplification oligonucleotide are coincident. Of course, amplification oligonucleotides in general also need to meet other requirements for attachment to a medium, such as inclusion of a group attached to the medium, etc. The requirement for partial matching is a common design approach.

According to a preferred embodiment, the template polynucleotide is hybridized to the surface of the medium by controlling the conditions such that the ratio of the number of single stranded template polynucleotides to the micro-reaction chamber in which the amplification oligonucleotides are planted or the microspheres in which the amplification oligonucleotides are planted is 1:1, such that most of the micro-reaction chambers hybridize to only one template sequence.

The nucleic acid templates are then amplified on the surface of the medium using an amplification technique, which according to a preferred embodiment is one of the amplification modes loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), recombinase-mediated isothermal nucleic acid amplification (RAA), nicking endonuclease isothermal amplification (NEAR), rolling Circle Amplification (RCA), nucleic acid sequence dependent amplification (NASBA), helicase Dependent Amplification (HDA), strand Displacement Amplification (SDA), bridge amplification or PCR.

According to a preferred embodiment, the amplification reagents described in the present invention may comprise a polymerase and dNTPs, as well as an amplification buffer. Alternatively, the amplification reagents may also include a recombinase, a single-stranded binding protein, and the like.

After amplification, the amplified oligonucleotides need to be sheared, capped, etc., to be sequenced later. Of the at least two amplification oligonucleotides on the surface of the medium, the first amplification oligonucleotide comprises a cleavage site and the second amplification oligonucleotide comprises a cleavage site. By acting on the cleavage site of the first amplification oligonucleotide, the second strand can be specifically cleaved off, and the first amplification oligonucleotide, which has not undergone an extension reaction, is also cleaved off. Then, the sheared sequence is unwound by adding an unwinding solution for reaction, and then a blocking solution is added, so that the 3' ends of all solid-phase polynucleotide chains or oligonucleotides are blocked and cannot be extended to generate a hetero signal under the action of DNA polymerase. After the above-described treatment steps, the first strand sequencing reaction can be performed by hybridizing the first sequencing primer.

In the present invention, the first amplification oligonucleotide comprises a cleavage site as close as possible to the 5' end of the oligonucleotide, so that the first amplification oligonucleotide is completely cleaved by the cleavage reaction to the maximum extent. The shearing reaction is combined with the subsequent end capping reaction, so that the occurrence of side reaction (nonspecific extension) in the sequencing process can be further reduced, and interference signals are reduced. Since the first amplification oligonucleotide is also required for the formation of the second strand, which is thoroughly sheared in this step, it is necessary to re-supply the amplification oligonucleotide into the reaction system prior to the formation of the second strand, which re-supply of amplification oligonucleotide comprises at least the first amplification oligonucleotide and in some embodiments may also provide the second amplification oligonucleotide.

According to a preferred embodiment, the two amplification oligonucleotides contain cleavage sites means that the cleavage sites on the two amplification oligonucleotides are different and the conditions of cleavage are also different. Thus, when shearing is performed, different shearing conditions are selected according to specific requirements, and favorable shearing can be controllably performed. Wherein, the shearing of the amplified double-stranded template into single strands refers to shearing the first amplification oligonucleotide or the second amplification oligonucleotide into two sections through a shearing site, so that the connection between the first amplification oligonucleotide or the second amplification oligonucleotide and the surface of the medium is disconnected.

In the present invention, after the shearing reaction is performed, it is necessary to subject the product of the shearing reaction to denaturing conditions to remove portions of the cleaved chains that are not attached to the surface of the medium. For example, the nucleic acid may be denatured by passing a formamide solution.

After shearing and capping, the first strand is present on the surface of the medium, the first sequencing primer is hybridized to the sequencing primer binding site on the first strand, and the first strand sequencing reaction is performed by sequentially adding nucleotides to the 3' end of the sequencing primer. The DNA polymerase uses sequencing primers to initiate synthesis and the single stranded polynucleotide serves as the first template, the result of the first strand sequencing reaction determining the sequence of the first strand and producing the complementary strand of the first strand.

According to a preferred embodiment, the sequencing is sequencing-by-synthesis or sequencing-by-ligation, preferably fluorescence-generated sequencing. The signal generation principle of fluorescence sequencing is that DNA polymerase combines a reaction substrate with a fluorescent group marked on 5' phosphate onto a sequencing template according to the base complementary pairing principle, and the released group can be further decomposed into a fluorogenic group under the action of alkaline phosphatase, so that the fluorescent group is released and emits fluorescence under the excitation light of a specific wavelength when the DNA template is extended. Since alkaline phosphatase is present during sequencing, the phosphate group remaining at the 3' end of the DNA after the cleavage reaction is removed prior to sequencing to prevent it from generating a hetero signal during sequencing.

In some preferred embodiments, after the first strand sequencing is complete, the single strand from the sequencing primer is unwound, the single strand is removed, the amplification oligonucleotide is provided again to the chip surface, and the amplification oligonucleotide is immobilized to the medium surface by the reaction, i.e., a solid phase amplification oligonucleotide is regenerated, comprising the first amplification oligonucleotide, with or without a cleavage site; optionally, the solid phase amplification oligonucleotide comprises a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of the first amplification oligonucleotide to the number of the second amplification oligonucleotide being any number between 1 and 10, preferably any number between 1 and 5; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site. Then, the first strand is hybridized with the first amplification oligonucleotide to provide an amplification reaction, amplification or extension is performed to generate a second strand immobilized on the surface of the medium, the second amplification oligonucleotide is sheared to remove the first strand and retain the second strand, a capping reaction is performed, and sequencing of the second strand can be performed after hybridization of the second sequencing primer. The amplification may be performed using isothermal amplification or conventional PCR.

In a preferred embodiment, the 5' end of the re-supplied amplification oligonucleotide of step (6) is modified with a specific group, immobilized on the surface of the support medium by reaction with a chemical group on the surface of the support medium, to form an intact amplification oligonucleotide, which is hybridized to the first strand; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto and the like. The reaction of a specific group modified on the amplification oligonucleotide with a group on the surface of the support medium may include, for example: the amino-modified amplification oligonucleotide may be attached to the surface of the carboxyl-modified support medium via NHS/EDC; the carboxyl-modified amplification oligonucleotide may be attached to the amino-modified support medium surface via NHS/EDC; an alkynyl modified amplification oligonucleotide can be click-linked to the azide group modified support medium surface; the azido group modified amplification oligonucleotide can be click-linked to the alkynyl modified support medium surface; and other commonly used chemical attachment methods. EDC/NHS can be used to graft amino and carboxyl groups, which are widely used because they do not introduce extra carbon chains in the chain, etc. EDC is a water-soluble carbodiimide, commonly used as an activating reagent for carboxyl groups, and is used with NHS at a pH ranging from 4.0 to 6.0 to improve coupling efficiency.

In some preferred embodiments, step (7) comprises providing an amplification reaction, performing amplification or extension, to generate a plurality of double-stranded polynucleotides immobilized on the surface of the medium, the double-stranded polynucleotides comprising a first strand and a second strand. When the number of first strands is sufficient, only the extension reaction may be performed, and a sufficient amount of second strands may be generated, and when the number of first strands is insufficient, the number of second strands may be further increased by amplification, in order to obtain a sequencing signal of sufficient intensity. The amplification is one of amplification modes such as loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), recombinase-mediated isothermal nucleic acid amplification (RAA), nicking endonuclease isothermal amplification (NEAR), rolling Circle Amplification (RCA), nucleic acid sequence dependent amplification (NASBA), helicase Dependent Amplification (HDA), strand Displacement Amplification (SDA), bridge amplification or PCR.

In some preferred embodiments, the re-provided amplification oligonucleotide of step (6) comprises a liquid phase first amplification oligonucleotide, which can hybridize to the first strand, the first amplification oligonucleotide being modified at the 5' end with a specific group; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto and the like.

According to a preferred embodiment, step (7) comprises hybridizing the liquid phase of the first amplification oligonucleotide to the first strand to provide an amplification reactant, extending a second strand; the second strand is immobilized to the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium.

According to a preferred embodiment, step (7) may further comprise hybridizing the liquid phase of the first amplification oligonucleotide to the first strand, immobilizing the first amplification oligonucleotide on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium; providing an amplification reaction, performing amplification or extension of the surface of the medium, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

According to a preferred embodiment, step (7) may further comprise providing a liquid phase amplification oligonucleotide modified at the 5' end with a specific group, immobilizing the liquid phase amplification oligonucleotide and the first amplification oligonucleotide hybridized to the first strand in step (6) on the surface of the support medium by reaction with a chemical group on the surface of the support medium; providing an amplification reactant, and performing medium surface amplification or extension to generate a second chain fixed on the surface of the medium; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, cycloalkynyl, maleimide, succinimide, mercapto and the like.

In the present invention, the re-provided first amplification oligonucleotide may or may not include a cleavage site. Since subsequent reactions do not require cleavage of the first amplification oligonucleotide, the absence of cleavage sites reduces the cost of primer synthesis and is also a preferred embodiment. It will be appreciated that when the first amplification oligonucleotide does not comprise a cleavage site, its nucleotide sequence composition is identical to that of the solid phase first amplification oligonucleotide which has been cleaved. The re-provided second amplification oligonucleotide comprises a cleavage site thereon and is identical to the cleavage site on the solid phase second amplification oligonucleotide on the surface of the medium.

In the present invention, a plurality of template polynucleotides, for example, 10 ⁵、10⁶、10⁷ or more template polynucleotides are included in the amplification reaction, and the number of template polynucleotides may be in the range of nanograms to micrograms.

The amplification method of the present invention is preferably performed on a solid phase. For example, a variety of amplification oligonucleotides for amplification are attached to the surface of a solid support medium.

In the present invention, it may be advantageous to add a cleaning solution for washing between the steps of the amplification sequencing method.

In some preferred embodiments, the mediator material is a hydrogel layer, such as a polyacrylamide hydrogel layer, which serves as an intermediate layer to which the amplification oligonucleotide may be covalently attached, and which may also be attached by non-covalent interactions to the inert substrate or matrix, including but not limited to glass, silicon wafer, etc.

In some preferred embodiments, the vehicle material is a hydrogel microsphere. The hydrogel microsphere is synthesized by chemical synthesis means, and then the reactive groups are reserved and react with the chemical groups on the amplified oligonucleotides, so that the amplified oligonucleotides are immobilized on the microsphere surface. As a disclosure, applicant's previous patents such as CN202010087598.8, e.g. CN202010986795.3, e.g. CN202010063461.9, describe methods and procedures for synthesizing hydrogels and the like types of microspheres.

In some embodiments, magnetic microspheres are also contemplated as media materials. The magnetic microsphere can exert acting force through an external magnetic field and can play a role under certain special conditions.

Schematic of the double-ended amplification sequencing process on the chip surface is shown in FIG. 1. The biotin carried on the microsphere can be combined with streptavidin on the surface of the chip, so that the microsphere can be firstly immobilized in a micro-pit on the surface of the chip in a centrifugal mode. Two solid phase primers (i.e., 101 and 102) are immobilized on the microsphere, and both primers have cleavage sites (i.e., 104 and 105) for subsequent cleavage steps, and the microsphere surface also has a specific group modification 103 (step (1) in the figure). Before the start of amplification, the single-stranded template polynucleotide is first hybridized to the microsphere (step (2) in the figure), followed by initial extension (step (3) in the figure), unwinding, and removal of the liquid phase template (step (4) in the figure). Then, amplification of the template is performed (step (5) and (6) in the figure). After the amplification is completed, the solid phase primer is subjected to a cleavage reaction (step (7) in the figure), a unwinding reaction (step (8) in the figure) and a blocking reaction (step (9) in the figure) in this order. At this time, the template on the microsphere is the first strand, and the first strand sequencing can be performed by hybridizing the first sequencing primer to the first strand (step (10) in the figure). After the sequencing is completed, the single strand extended from the sequencing primer is unwound, the first amplification oligonucleotide is regenerated on the microsphere surface by reaction (step (11) in the figure), and then the isothermal amplification or PCR is used for regenerative extension or amplification (steps (12) (13) in the figure). After amplification is complete, another cleavage solution is added to cleave the first strand at the cleavage site on the second amplification oligonucleotide (step (14) of the figure), and a helicase reaction is added to unwind the cleaved sequence, exposing a single strand that can bind to the sequencing primer (step (15) of the figure). A blocking solution was also added to the chip so that the 3' -end of all solid phase DNA was blocked (step (16) in the figure). Finally, after the above-mentioned treatment step, the second strand can be sequenced by hybridizing the second sequencing primer (step (17) in the figure).

In general sequencing, the DNA molecule of the fragment is in a double-stranded form, and it is necessary to unwind the double-stranded molecule into a single-stranded molecule before hybridization. The template polynucleotide is a double-stranded nucleic acid library or a single-stranded nucleic acid library, and before hybridization, double-stranded DNA needs to be unwound into single strands, namely, single-stranded template polynucleotide is provided and then is complementarily paired with the solid-phase amplification oligonucleotide.

Preferably, the template polynucleotide comprises a first index and a second index to distinguish the source of the nucleic acid sequence. The double-ended amplification sequencing method of the present invention further comprises sequencing the first index and the second index. For example, the first chain may be measured first, the first index may be measured second, the second chain may be measured second, and the second index may be measured second; or first measuring a first index, then measuring a first chain, then measuring a second chain and then measuring a second index; or first measuring a first index, then measuring a first chain, then measuring a second index and then measuring a second chain; or first measuring a first chain, then measuring a first index, then measuring a second index and then measuring a second chain; the sequencing order of the index sequence is adjustable and can be selected according to the actual sequencing.

The template polynucleotide of the present invention refers to any nucleic acid fragment sequence. During high throughput sequencing, a large number of nucleic acid fragments, e.g., in excess of 100M, are present at a time, any one of which may be referred to as a template polynucleotide.

For gene sequencing methods, conventional, e.g., illumina, measure one base per round. Most sequencing methods measure a single base signal at a time. It is not essential to the invention that a few bases are reacted at a time. The double-end amplification sequencing method disclosed by the invention is more suitable for sequencing of 2+2 fluorescence switching types disclosed by the applicant before; the method of the present invention exhibits very strong compatibility, but does not exclude conventional sequencing methods similar to Illumina type. In particular, the content of patent cn2015110815685. X can be incorporated into this patent by reference.

The biochemical compounds or modifications to which the present invention relates are commercially available unless otherwise specified.

Example 1

1. Immobilizing the microsphere on the surface of the chip, wherein the microsphere surface is modified with an immobilization group and two amplification primers (namely a first amplification primer and a second amplification primer):

1) The microsphere carrier was diluted to about 3M/. Mu.L with a1 xPS solution containing 0.01% Tween20 and mixed by shaking.

2) And 300 mu L of diluted microsphere solution is introduced into a sample inlet of the chip.

3) The chip was centrifuged.

2. Template prehybridization

1) Depending on the final template concentration added by the amplification reaction, the template is further diluted and a suitable volume of 0.1M NaOH is added for de-spinning. The template is diluted and de-screwed to obtain a 1.5ml DNA Lobind EP pipe; if the diluted template does not need to be stored for a long time, the diluted template can be diluted by using ultra water, and if the diluted template needs to be stored for a long time, the diluted template can be diluted by using TE. The following table shows the usual template concentration and the volume of 0.1M NaOH added.

Final concentration of template	Template added and 0.1M NaOH volume
		1pM	4 Mu L of 100pM template+4 mu L of NaOH

2) After adding 0.1M NaOH, shaking and mixing uniformly, rapidly centrifuging for 2s, and standing at room temperature for 5min.

3) After the unwinding is finished, adding 5 XSSC solution according to the volume of the unwinding liquid to make the final volume of the solution 400 mu L, slowly blowing the solution by a pipetting gun for 10 times, uniformly mixing, immediately placing the solution on ice for later use, and recording that vibration and uniform mixing cannot be performed.

4) The chip to be amplified is cleaned by 500 mu L of seq buffer, if bubbles exist, 200 mu L of IPA can be added to drive the bubbles, and then the chip to be amplified is cleaned by 500 mu L of seq buffer.

5) Adding template solution into chips, adding 200 mu L of reaction solution into each chip, wiping the liquid at the inlet and outlet of the chip with kimtech, sealing with sealing film, placing into a flat-plate PCR instrument, and selecting an ANNEAL program: 96 ℃ for 30s; -0.05 ℃/s;40 ℃ for 10s;25℃for forever.

6) Taking out after the reaction is finished, and placing on ice for standby.

3. Initial extension reaction

1) 400 Mu L of 2x phusion mixed solution is taken and placed on ice, 400 mu L of ultrapure water is added, diluted by one time, and the mixture is vibrated and mixed uniformly and placed on ice.

2) The above mixture was added to each chip in an amount of 400. Mu.L, and the chips were placed on a plate PCR apparatus and heated at 72℃for 2 minutes.

3) After the reaction is finished, 400 mu L of formamide is added into the chip, and the chip is cleaned by a cleaning solution after the reaction is carried out for 5min at normal temperature.

4. Recombinase polymerase amplification reaction

1) The preparation of the reaction solution was carried out according to the following table.

2) After the preparation is completed, the mixture is vibrated and mixed evenly, and the mixture is rapidly centrifuged for 2s.

3) The chip is cleaned with 500. Mu.L of cleaning solution, and if bubbles exist, 200. Mu.L of isopropanol can be added to remove bubbles, and then 500. Mu.L of cleaning solution is used for cleaning.

4) Adding the prepared and mixed amplification reaction solution into a chip, placing the chip on a PCR instrument, screwing a cover, and selecting a heating program: 40 ℃ for 60min;4℃for forever.

5. Shearing (USER enzyme mix) reaction, shearing the first amplification primer

1) A1.5 ml DNA Lobind EP-tube was used to prepare a shear reaction solution according to the following table.

Reagent(s)	V/μL
		USER enzyme	4
Cutsmart	40
		ultrapure water	356
Total	400

2) After the preparation is completed, the materials are vibrated and mixed uniformly, and are rapidly centrifuged for 2s and placed on ice for standby.

3) The chip after the amplification reaction is washed with 500. Mu.L of a washing liquid, and if bubbles are present, 200. Mu.L of IPA may be added to remove bubbles and then washed with 500. Mu.L of a washing liquid.

4) Adding the prepared and mixed shearing reaction liquid into a chip, placing the chip on a flat-plate PCR instrument, screwing a cover, and selecting a shearing reaction program: 37 ℃ for 30min;4℃for forever.

6. Unwinding

1) The chip after the reaction of the shearing solution is cleaned by 500. Mu.L of cleaning solution, and if bubbles exist, 200. Mu.L of IPA can be added to remove bubbles, and then 500. Mu.L of cleaning solution can be used for cleaning.

2) 200. Mu.L of formamide was added to each chip and reacted at room temperature (25 ℃) for 10min.

3) After the reaction was completed, 1ml of 1xTE was added for washing, and if not immediately sequenced, the reaction was sealed with a sealing film and stored in a storage box of a refrigerator at 4 ℃.

7. End-capping solution (TdT enzyme mix) reaction

1) 1.5Ml DNA Lobind EP tubes were used to prepare the end-capping reaction solutions according to the following table.

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3) The chip is cleaned with 500. Mu.L of cleaning solution, and if bubbles are present, 200. Mu.L of IPA can be added to remove bubbles and then the chip is cleaned with 500. Mu.L of cleaning solution.

4) Adding the prepared and mixed end-capping reaction solution into a chip, placing the chip on a flat-plate PCR instrument, screwing a cover, and selecting an end-capping reaction program: 37 ℃ for 30min;4℃for forever.

8. First strand sequencing

1) The sequencing primers dissolved in 1 xTE at a concentration of 100. Mu.M were diluted to 2. Mu.M with hybridization solution in EP tube.

3) The end-capped chip is cleaned with 500. Mu.L of cleaning solution, and if bubbles are present, 200. Mu.L of IPA can be added to remove bubbles and then cleaned with 500. Mu.L of hybridization solution.

4) Adding the prepared and mixed sequencing primer solution into a chip, placing the chip on a flat-plate PCR instrument, screwing a cover, and selecting a sequencing primer hybridization program: 60 ℃ for 7min;40℃for 3min.

5) After the completion of the reaction, 500. Mu.L of a cleaning liquid was added thereto for cleaning. A first round of sequencing was then performed.

9. Re-hybridizing the first amplification primer

1) The first amplification primer dissolved in 1 XTE at a concentration of 100uM was diluted to 1uM in the EP tube with the hybridization solution.

2) After the preparation is completed, the mixture is vibrated and evenly mixed, and the mixture is rapidly centrifuged for 2 seconds.

3) The chip is cleaned by 500ul of cleaning solution, if bubbles exist in the chip, 1ml of IPA can be added to remove bubbles, and then 1ml of cleaning solution is used for cleaning.

4) 500Ul of primer solution was added to the chip, after sealing, the chip was placed on a plate PCR instrument, the lid was screwed down, and the primer hybridization procedure was selected: 60 ℃,7min,40 ℃ and 3min.

5) After the reaction was completed, 1ml of a cleaning liquid was added for cleaning.

10. Generating a second chain

1) An extension reaction solution was prepared by taking 1.5ml of an EP tube according to the following system.

Reagent(s)	V/ul
		Hydration liquid	180
Magnesium acetate	15
		Ultrapure water	105
Total volume of	300

2) The chip was cleaned with 1ml of cleaning solution, and if there were bubbles, 1ml of IPA was added to remove bubbles.

3) Adding 300ul of extension reaction solution into the chip, sealing, placing the chip on a flat-plate PCR instrument, and tightening

PCR instrument lid, select extension procedure: 40 ℃,20min,4 ℃ for forever.

4) After the reaction was completed, 1ml of a cleaning liquid was added to the chip to clean the chip.

11. Solid phase ligation reaction

1) A1.5 ml DNA Lobind EP-tube was used to prepare a solid phase ligation reaction solution according to the following table.

Reagent(s)	V/ul
		B3T	283
Catalyst	17
		Total	300

3) The reacted chip is cleaned with 500ul of cleaning solution, if bubbles exist, 200ul of IPA can be added to remove bubbles, and then 500ul of cleaning solution is used for cleaning.

4) And adding the prepared and mixed reaction solution into a chip, and standing for 1h at room temperature.

12. Shearing (Fpg enzyme mix) reaction, shearing the second amplification primer

Reagent(s)	V/μL
		Fpg enzyme	10
10x NEB buffer	40
		BSA	4
ultrapure water	346
		Total	400

2) The chip was rinsed with 1ml of rinse solution and if there were bubbles, 1ml of IPA was used to drive the bubbles out.

3) Adding the shearing reaction liquid into a chip, sealing, placing on a flat-plate PCR instrument, and selecting a shearing program: 37℃for 1h.

4) After the shearing was completed, the chip was washed with 1ml of a washing liquid. If there is a bubble, the bubble can be removed with 1ml IPA.

5) 1Ml of the unwinding liquid was added to the chip, and the chip was left at room temperature for 3min to unwind.

6) 1Ml of the washing liquid was added to wash the chip. If there is a bubble, the bubble can be removed with 1ml IPA.

13. Reaction of cutting phosphoric acid

1) A1.5 ml EP tube was used to prepare a phosphorus-containing acid reaction solution according to the following system.

Reagent(s)	V/ul
		T4PNK enzyme	16
10x NEB buffer	40
		Ultrapure water	344
Total	400

3) Adding the phosphoric acid cutting reaction liquid into a chip, sealing, placing on a flat-plate PCR instrument, and selecting a shearing program: 37℃for 1h.

14. Capping reactions

Reagent(s)	V/μL
		TdT enzyme	4
10x buffer	40
		CoCl₂	40
dTTP	4
		ultrapure water	312
Total	400

15. Second strand sequencing

3) The chip after reaction is washed with 500. Mu.L of a washing liquid, and if bubbles are present, 200. Mu.L of IPA may be added to remove bubbles and then washed with 500. Mu.L of a hybridization solution.

5) After the completion of the reaction, 500. Mu.L of a cleaning liquid was added thereto for cleaning. Second strand sequencing was then performed.

16. Data analysis

Second strand generation efficiency:

The left graph of FIG. 2A shows the brightness of the microsphere when the fluorescent labeled probe is hybridized after the first strand is generated, and the right graph of FIG. 2A shows the brightness of the microsphere when the newly generated second strand product is hybridized with the fluorescent labeled probe, and the brightness of the microsphere can reflect the quantity of the corresponding products because the probes used by the first strand and the second strand are labeled by the same fluorescein. FIG. 2B is a graph of second strand generation efficiency plotted against microsphere brightness using MATLAB software, wherein the X-axis is microsphere brightness when hybridized to a first strand fluorescently labeled probe and the Y-axis is microsphere brightness when hybridized to a second strand fluorescently labeled probe, and the slope of the scatter plot indicates second strand generation efficiency. From the figure, the second strand generation efficiency is 80% of that of the first strand, and the second strand generation efficiency is higher, meeting the second strand sequencing requirement.

Example 2

Steps 1-8 are the same as in example 1, supra.

9. Re-hybridizing the first amplification primer

10. Solid phase ligation reaction

Reagent(s)	V/ul
		B3T	283
Catalyst	17
		Total	300

11. Generating a second chain

3) Adding 300ul of extension reaction liquid into the chip, sealing, placing the chip on a flat-plate PCR instrument, screwing the cover of the PCR instrument, and selecting an extension program: 40 ℃,20min,4 ℃ for forever.

7) A1.5 ml DNA Lobind EP-tube was used to prepare a shear reaction solution according to the following table.

8) The chip was rinsed with 1ml of rinse solution and if there were bubbles, 1ml of IPA was used to drive the bubbles out.

9) Adding the shearing reaction liquid into a chip, sealing, placing on a flat-plate PCR instrument, and selecting a shearing program: 37℃for 1h.

10 After the shearing was completed, the chip was washed with 1ml of a washing liquid. If there is a bubble, the bubble can be removed with 1ml IPA.

111 Ml of the unwinding liquid was added to the chip, and the chip was left at room temperature for 3min, and the unwinding was performed.

12 1Ml of the cleaning liquid was added to clean the chip. If there is a bubble, the bubble can be removed with 1ml IPA.

13. Reaction of cutting phosphoric acid

4) A1.5 ml EP tube was used to prepare a phosphorus-containing acid reaction solution according to the following system.

Reagent(s)	V/ul
		T4PNK enzyme	16
10x NEB buffer	40
		Ultrapure water	344
Total	400

5) The chip was rinsed with 1ml of rinse solution and if there were bubbles, 1ml of IPA was used to drive the bubbles out.

6) Adding the phosphoric acid cutting reaction liquid into a chip, sealing, placing on a flat-plate PCR instrument, and selecting a shearing program: 37℃for 1h.

14. Capping reactions

15. Second strand sequencing

16. Data analysis

Second strand generation efficiency:

The left graph of FIG. 3A shows the brightness of the microsphere when the fluorescent labeled probe is hybridized after the first strand is generated, and the right graph of FIG. 3A shows the brightness of the microsphere when the newly generated second strand product is hybridized with the fluorescent labeled probe, and the brightness of the microsphere can reflect the quantity of the corresponding products because the probes used by the first strand and the second strand are labeled by the same fluorescein. FIG. 3B is a graph of second strand generation efficiency plotted against microsphere brightness using MATLAB software, wherein the X-axis is microsphere brightness when hybridized to a first strand fluorescently labeled probe and the Y-axis is microsphere brightness when hybridized to a second strand fluorescently labeled probe, and the slope of the scatter plot indicates second strand generation efficiency. From the figure, the second strand generation efficiency is 88% of that of the first strand, and the second strand generation efficiency is higher, meeting the second strand sequencing requirement.

Example 3

Steps 1-8 are the same as in example 1, supra.

9. Unwinding

1) The sequenced chip was cleaned with 500. Mu.L of cleaning solution, and if bubbles were present, 200. Mu.L of IPA was added to drive off bubbles and then with 500. Mu.L of cleaning solution.

3) After the completion of the reaction, 1ml of 1xTE was added thereto for washing.

10. Solid phase ligation of first amplification primer

1) The first amplification primer was dissolved in 1 xTE to give a final concentration of 100. Mu.M.

2) A1.5 ml DNA Lobind EP-tube was used to prepare a solid phase ligation reaction solution according to the following table.

Reagent(s)	V/ul
		Amplification primers	2
B3T	281
		Catalyst	17
Total	300

3) After the preparation is completed, the mixture is vibrated and mixed evenly, and the mixture is rapidly centrifuged for 2s.

4) The reacted chip is cleaned with 500ul of cleaning solution, if bubbles exist, 200ul of IPA can be added to remove bubbles, and then 500ul of cleaning solution is used for cleaning.

5) And adding the prepared and mixed reaction solution into a chip, and standing for 1h at room temperature.

11. Second Strand Forming reaction

13. Reaction of cutting phosphoric acid

Reagent(s)	V/ul
		T4PNK enzyme	16
10x NEB buffer	40
		Ultrapure water	344
Total	400

14. Capping reactions

15. Second strand sequencing

16. Data analysis

Second strand generation efficiency:

The left graph of FIG. 4A shows the brightness of the microsphere when the fluorescent labeled probe is hybridized after the first strand is generated, and the right graph of FIG. 4A shows the brightness of the microsphere when the newly generated second strand product is hybridized with the fluorescent labeled probe, and the brightness of the microsphere can reflect the quantity of the corresponding products because the probes used by the first strand and the second strand are labeled by the same fluorescein. FIG. 4B is a graph of second strand generation efficiency plotted against microsphere brightness using MATLAB software, wherein the X-axis is microsphere brightness when hybridized to a first strand fluorescently labeled probe and the Y-axis is microsphere brightness when hybridized to a second strand fluorescently labeled probe, and the slope of the scatter plot indicates second strand generation efficiency. From the figure, the second strand generation efficiency is 79% of the first strand, and the second strand generation efficiency is higher, meeting the second strand sequencing requirement.

Example 4

Steps 1-8 are the same as in example 1, supra.

9. Re-hybridizing the first amplification primer

10. Solid phase ligation of first amplification primer

Reagent(s)	V/ul
		Amplification primers	1
B3T	282
		Catalyst	17
Total	300

11. Second Strand Forming reaction

13. Reaction of cutting phosphoric acid

Reagent(s)	V/ul
		T4PNK enzyme	16
10x NEB buffer	40
		Ultrapure water	344
Total	400

14. Capping reactions

15. Second strand sequencing

16. Data analysis

Second strand generation efficiency:

The left graph of FIG. 5A shows the brightness of the microsphere when the first strand is hybridized with the fluorescent label probe, and the right graph of FIG. 5A shows the brightness of the microsphere when the newly generated second strand product is hybridized with the fluorescent label probe, and the brightness of the microsphere can reflect the quantity of the corresponding products because the probes used by the first strand and the second strand are labeled by the same fluorescein. FIG. 5B is a graph of second strand generation efficiency plotted against microsphere brightness using MATLAB software, wherein the X-axis is microsphere brightness when hybridized to a first strand fluorescently labeled probe and the Y-axis is microsphere brightness when hybridized to a second strand fluorescently labeled probe, and the slope of the scatter plot indicates second strand generation efficiency. From the figure, the second strand generation efficiency is 77% of the first strand, and the second strand generation efficiency is higher, meeting the second strand sequencing requirement.

The foregoing discloses specific embodiments and details of the overall process. The present invention is not limited to the above-described embodiments, but may be modified or altered by persons skilled in the art based on the description.

Claims

1. A method for double-ended amplification sequencing of a chip surface, comprising:

2. The method of claim 1, wherein the support medium surface has discrete depressions in the shape of a cylinder, a truncated cone, a groove, a truncated cone, a hexagonal column, or a combination thereof, for the reaction of the microreactor.

3. The method of claim 1, wherein the support medium is an inert substrate or matrix, the material of which includes, but is not limited to, glass, silicon, optical fiber, resin, ceramic, metal, nitrocellulose, polyethylene, polystyrene, copolymers of styrene and other materials, polypropylene, acrylic, polybutylene, or polyurethane.

4. The method of claim 1, wherein the support medium comprises an inert substrate or matrix and a mediator material directly attached to the amplification oligonucleotide and linked to the inert substrate or matrix by covalent or non-covalent forces; the medium materials include, but are not limited to, hydrogel layers, hydrogel microspheres, and magnetic microspheres; materials for the inert substrate or matrix include, but are not limited to, glass, silicon, optical fibers, resins, ceramics, metals, nitrocellulose, polyethylene, polystyrene, copolymers of styrene with other materials, polypropylene, acrylic, polybutylene, or polyurethane.

5. The method of claim 1, wherein the chemical groups supporting surface modification of the medium comprise amino, carboxyl, epoxy, hydroxyl, aldehyde, azide, alkyne, maleimide, succinimide, or sulfhydryl groups; the amplification oligonucleotide is immobilized on the surface of the support medium by reaction with the chemical group.

6. The method of claim 1, wherein the chemical groups that support medium surface modifications comprise cyclic alkyne groups, and the amplification oligonucleotide is immobilized on the support medium surface by reaction with the chemical groups.

7. The method of claim 1, wherein the cleavage site allows enzymatic cleavage, chemical cleavage or photochemical cleavage.

8. The method of claim 7, wherein the cleavage site comprises a site cleaved with a nicking endonuclease.

9. The method of claim 7, wherein the shearing comprises contacting the support medium surface with a composition comprising at least one enzyme to create abasic sites at the shearing sites, wherein the shearing occurs at the shearing sites.

10. The method of claim 9, wherein the amplification oligonucleotide comprises a uracil base or an 8-oxoguanine base or a deoxyhypoxanthine base.

11. The method of claim 9, wherein the amplification oligonucleotide comprises tetrahydrofuran modified bases.

12. The method of any one of claims 9-11, wherein the at least one enzyme that produces an abasic site at the cleavage site comprises uracil DNA glycosylase and an endonuclease or endonuclease iv selected from the group consisting of DNA glycosylase-lyase endonucleases viii or Fpg glycosylase.

13. The method of claim 1, wherein the cleavage site is selected from the group consisting of uracil bases, 8-oxoguanine bases, deoxyinosine bases, ortho-dihydroxyl modified phosphoramidite sites, disulfide bonds, azo groups, azide groups, peptide bonds, ketals, acetals, diphenylsiloxanes, carbonates, and carbamates.

14. The method of claim 1, wherein the cleavage site is a tetrahydrofuran modified base.

15. The method of claim 1, wherein the cleavage site is one or more ribonucleotides.

16. The method of claim 1, wherein the amplification is one of loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), recombinase-mediated isothermal nucleic acid amplification (RAA), nicking endonuclease isothermal amplification (NEAR), rolling Circle Amplification (RCA), nucleic acid sequence dependent amplification (NASBA), helicase Dependent Amplification (HDA), strand Displacement Amplification (SDA), or bridge PCR.

17. The method of claim 1, wherein the 5' end of the re-supplied amplification oligonucleotide in step (6) is modified with a specific group, and is immobilized on the surface of the support medium by reaction with a chemical group on the surface of the support medium to form a complete amplification oligonucleotide, which is hybridized to the first strand; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, maleimide, succinimide or mercapto.

18. The method of claim 17, wherein the re-provided amplification oligonucleotides comprise a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotides to the number of second amplification oligonucleotides being any number between 1-10; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site.

19. The method of claim 17, wherein the re-provided amplification oligonucleotides comprise a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotides to the number of second amplification oligonucleotides being any number between 1-5; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site.

20. The method of claim 17, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

21. The method of claim 18 or 19, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

22. The method of claim 1, wherein the 5' end of the re-supplied amplification oligonucleotide in step (6) is modified with a specific group, and is immobilized on the surface of the support medium by reaction with a chemical group on the surface of the support medium to form a complete amplification oligonucleotide, which is hybridized to the first strand; the specific group is a cycloalkynyl group.

23. The method of claim 22, wherein the re-provided amplification oligonucleotides comprise a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotides to the number of second amplification oligonucleotides being any number between 1-10; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site.

24. The method of claim 22, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

25. The method of claim 23, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

26. The method according to claim 1, wherein the 5' end of the re-supplied amplification oligonucleotide in step (6) is modified with a specific group to be immobilized on the surface of the support medium by reacting with a chemical group on the surface of the support medium; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, maleimide, succinimide or mercapto.

27. The method of claim 26, wherein the re-provided amplification oligonucleotides comprise a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotides to the number of second amplification oligonucleotides being any number between 1-10; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site.

28. The method of claim 26, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

29. The method of claim 27, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

30. The method according to claim 1, wherein the 5' end of the re-supplied amplification oligonucleotide in step (6) is modified with a specific group to be immobilized on the surface of the support medium by reacting with a chemical group on the surface of the support medium; the specific group is a cycloalkynyl group.

31. The method of claim 30, wherein the re-provided amplification oligonucleotides comprise a first amplification oligonucleotide and a second amplification oligonucleotide, the ratio of the number of first amplification oligonucleotides to the number of second amplification oligonucleotides being any number between 1-10; the re-provided first amplification oligonucleotide comprises a cleavage site or does not comprise a cleavage site; the re-provided second amplification oligonucleotide comprises a cleavage site.

32. The method of claim 30, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

33. The method of claim 31, wherein step (7) comprises providing amplification reagents, performing amplification or extension, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

34. The method of claim 1, wherein the re-provided amplification oligonucleotide of step (6) comprises a liquid phase first amplification oligonucleotide capable of hybridizing to the first strand, the first amplification oligonucleotide being modified at the 5' end with a specific group; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, maleimide, succinimide or mercapto.

35. The method of claim 34, wherein step (7) comprises hybridizing the liquid phase first amplification oligonucleotide to the first strand to provide an amplification reactant, extending a second strand; the second strand is immobilized on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium.

36. The method of claim 1, wherein the re-provided amplification oligonucleotide of step (6) comprises a liquid phase first amplification oligonucleotide capable of hybridizing to the first strand, the first amplification oligonucleotide being modified at the 5' end with a specific group; the specific group is a cycloalkynyl group.

37. The method of claim 36, wherein step (7) comprises hybridizing the liquid phase first amplification oligonucleotide to the first strand to provide an amplification reactant, extending a second strand; the second strand is immobilized on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium.

38. The method of claim 34, wherein step (7) comprises hybridizing the first amplification oligonucleotide in the liquid phase to the first strand, and immobilizing the first amplification oligonucleotide on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium; providing an amplification reaction, performing amplification or extension of the surface of the medium, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

39. The method of claim 36, wherein step (7) comprises hybridizing the first amplification oligonucleotide in the liquid phase to the first strand, and immobilizing the first amplification oligonucleotide on the surface of the support medium by reacting a specific group at the 5' end of the first amplification oligonucleotide with a chemical group on the surface of the support medium; providing an amplification reaction, performing amplification or extension of the surface of the medium, and generating a second strand complementary to the first strand immobilized on the surface of the medium.

40. The method of claim 34, wherein step (7) comprises providing a liquid phase amplification oligonucleotide modified at the 5' end with a specific group, immobilizing said liquid phase amplification oligonucleotide and said first amplification oligonucleotide hybridized to said first strand in step (6) on the surface of a support medium by reaction with a chemical group on the surface of the support medium; providing an amplification reactant, and performing medium surface amplification or extension to generate a second chain fixed on the surface of the medium; the specific group is one or more of amino, carboxyl, epoxy, hydroxyl, aldehyde, azido, alkynyl, maleimide, succinimide or mercapto.

41. The method of claim 40, wherein the liquid phase amplification oligonucleotides modified with specific groups at the 5' end provided in step (7) comprise at least a first amplification oligonucleotide and optionally a second amplification oligonucleotide in a number ratio of any number between 1 and 10; the re-provided first amplification oligonucleotide comprises or does not comprise a cleavage site and the re-provided second amplification oligonucleotide comprises a cleavage site thereon.

42. The method of claim 40, wherein the liquid phase amplification oligonucleotides modified with specific groups at the 5' end provided in step (7) comprise at least a first amplification oligonucleotide and optionally a second amplification oligonucleotide in a number ratio of any number between 1 and 5; the re-provided first amplification oligonucleotide comprises or does not comprise a cleavage site and the re-provided second amplification oligonucleotide comprises a cleavage site thereon.

43. The method of claim 36, wherein step (7) comprises providing a liquid phase amplification oligonucleotide modified at the 5' end with a specified group, immobilizing said liquid phase amplification oligonucleotide and said first amplification oligonucleotide hybridized to said first strand in step (6) on the surface of a support medium by reaction with a chemical group on the surface of the support medium; providing an amplification reactant, and performing medium surface amplification or extension to generate a second chain fixed on the surface of the medium; the specific group is a cycloalkynyl group.

44. The method of claim 43, wherein the liquid phase amplification oligonucleotide modified with a specific group at the 5' end provided in step (7) comprises at least a first amplification oligonucleotide and optionally a second amplification oligonucleotide in a ratio of 1 to 10; the re-provided first amplification oligonucleotide comprises or does not comprise a cleavage site and the re-provided second amplification oligonucleotide comprises a cleavage site thereon.

45. The method of claim 43, wherein the liquid phase amplification oligonucleotide modified with a specific group at the 5' end provided in step (7) comprises at least a first amplification oligonucleotide and optionally a second amplification oligonucleotide in a ratio of any number between 1 and 5; the re-provided first amplification oligonucleotide comprises or does not comprise a cleavage site and the re-provided second amplification oligonucleotide comprises a cleavage site thereon.

46. The method of claim 1, wherein the template polynucleotide comprises a first index and a second index.

47. The method of claim 46, further comprising sequencing the first index and the second index.

48. The method of any one of claims 1-11 or 13-20 or 22-47, wherein the sequencing is sequencing-by-synthesis or sequencing-by-ligation.

49. The method of any one of claims 1-11 or 13-20 or 22-47, wherein the sequencing is fluorescence sequencing.

50. The method of claim 12, wherein the sequencing is sequencing-by-synthesis or sequencing-by-ligation.

51. The method of claim 12, wherein the sequencing is fluorescence sequencing.

52. The method of claim 21, wherein the sequencing is sequencing-by-synthesis or sequencing-by-ligation.

53. The method of claim 21, wherein the sequencing is fluorescence sequencing.