A template polynucleotide paired end sequencing method and kit

Abstract

The present invention relates to the field of gene sequencing technology, and in particular to a template polynucleotide paired end sequencing method and kit. The present invention specially designs an extension primer for generating a complementary chain to form an extension primer structure that is at least connected as a whole by a first extension primer and a second extension primer, and designs the binding site of the first extension primer on the template polynucleotide to be located upstream of the second extension primer binding site. In an environment with chain displacement activity, the extension chain of the upstream extension primer displaces the extension chain of the downstream extension primer, and the displaced extension chain of the downstream extension primer is connected to the first extension primer through the second extension primer, and then bound to the template polynucleotide chain through the extension chain of the first extension primer, and then fixed on a solid support through the template polynucleotide chain, thereby improving the stability of the complementary chain in a cycle sequencing environment and improving the sequencing quality.

Description

Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and in particular to a template polynucleotide paired-end sequencing method and a kit. Background Art

[0002] Paired-end or paired sequencing technology is well known in the field of analytical biology. Paired-end sequencing allows the determination of two reads of sequences from two locations on a polynucleotide double strand. Since paired-end sequences are not completely random, but are known to occur on a single double strand and are connected or paired in the genome, it helps to combine the sequences of the entire genome into a consistent sequence and increase the length of the sequence information obtained in one sequencing.

[0003] The paired-end sequencing methods based on array polynucleotide templates disclosed in the prior art include the method disclosed in WO2004 / 070005 and implemented on a solid support, wherein two or more primers are hybridized to the target polynucleotide at the same time, and after the hybridization step, all primers hybridized to the template except one primer are blocked, and the unblocked primers are extended for sequencing, and after the sequencing is completed, one of the blocked primers is unblocked to obtain a free 3' hydroxyl group for another extended sequencing reaction. During the implementation of this method, although the array polynucleotide template is always connected to the solid support to maintain the stability of the polynucleotide template chain, it is necessary to ensure that only one primer can be extended, and the other primers must be completely blocked. In actual operation, it is difficult to ensure that the other primers are completely blocked, resulting in a high sequencing error rate;

[0004] WO2007010252 discloses forming a double-stranded template cluster on a solid support, denaturing the double-stranded template cluster to form a single-stranded cluster, removing one of the strands of a portion of the double-stranded template cluster, and sequencing the remaining strand; after completing one sequencing, removing the single-stranded template with the same sequence as that in the first sequencing in the remaining portion of the double-stranded template cluster, sequencing the remaining strand complementary to the removed template strand, and completing double-end sequencing of the double-stranded template. This method can only sequence one strand of a portion of the clusters at a time, and cannot fully utilize all the clusters, resulting in the sequencing signal strength not meeting expectations;

[0005] WO2008041002 discloses that one of the chains of all double-stranded template clusters is first removed, the remaining chain is sequenced, and then the remaining chain is used as a template for amplification to form its complementary chain, and the complementary chain is sequenced, and finally double-end sequencing is achieved; and specifically discloses that the formation of the complementary chain depends on the adapter primers provided on the solid support template that are complementary hybridized with the 3 ends of the first sequencing chain, and the method also depends on the formation of the double-stranded template cluster first, which limits the scope of application of the method. Therefore, it is necessary to improve the generation method of the complementary chain so that the implementation and application of the method does not depend on the double-stranded template cluster, nor on the special adapters specially provided on the solid support, so as to expand the scope of application of the double-end sequencing method.

[0006] For example, WO2016 / 133764 discloses a method for generating complementary chains based on the chain displacement principle to achieve double-end sequencing, wherein a second chain complementary to the template chain is formed by hybridizing and extending the first primer with the template chain, and the second primer is hybridized and extended with the template chain to form a third chain while displacing the second chain, controlling the displacement of the second chain, ensuring that the second chain is partially hybridized with the template chain at the 3 ends, forming a partially hybridized and partially overhanging second chain, and sequencing the overhanging part of the second chain. During the implementation of this method, the formed complementary chain (second chain) is fixed by partially hybridizing the second chain near the 3 ends with the template chain. The stability of this fixing method is relatively poor, and it is easy to fall off during the repeated cycle washing process of extended sequencing, reducing the sequencing signal and affecting the sequencing quality. In view of this, it is necessary to further improve the fixing method of the generated complementary chain to improve the stability of the complementary chain in the cycle sequencing reaction. Summary of the invention

[0007] One of the purposes of the present invention is to provide a method for paired-end sequencing of a polynucleotide template, thereby improving the stability of the complementary chain formed during the second-end sequencing process and improving the sequencing quality.

[0008] At the same time, the present invention also provides a kit for implementing the sequencing method of the present invention.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A template polynucleotide paired-end sequencing method comprises the following steps:

[0011] S1. providing a single-stranded template polynucleotide cluster in an array fixed on a solid support, adding a first sequencing primer, sequencing at least a portion of the sequence of the single-stranded template polynucleotide, and obtaining an extended polynucleotide chain of the first sequencing primer and corresponding first terminal sequence information;

[0012] S2, removing the extended polynucleotide chain of the first sequencing primer, and adding an extension primer; wherein the extension primer includes a first extension primer and a second extension primer connected as one, and the template polynucleotide has a binding site for the first extension primer and a binding site for the second extension primer; wherein the binding site of the first extension primer is located upstream of the binding site of the second extension primer;

[0013] S3, annealing and hybridizing the extension primer and the template polynucleotide, wherein the first extension chain generated by the first extension primer and complementary to the template polynucleotide at least partially replaces the second extension chain generated by the second extension primer and complementary to the template polynucleotide under an environment having chain displacement reaction activity, and the second extension chain has at least a portion that is not hybridized and bound to the template polynucleotide, which is called the overhang portion of the second extension chain;

[0014] S4. Add a second sequencing primer to sequence the overhanging portion of the second extended chain to obtain the corresponding second terminal sequence information, thereby completing paired-end sequencing of the template polynucleotide.

[0015] The overall scheme for achieving paired end sequencing generally involves first completing at least a portion of the sequencing of the single-stranded template polynucleotide to obtain the sequence information of the first end; then generating a complementary chain of the single-stranded template polynucleotide for sequencing to obtain the sequence information of the second end. However, the most important problem in the implementation of this overall scheme is how the generated complementary chain can be stably present in the environment of cyclic sequencing and flushing, that is, the most important problem is how to stably fix the generated complementary chain on a solid support. In order to solve the above problems, the present invention specially designs an extension primer for complementary hybridization extension with the template polynucleotide to generate a complementary chain, forming an extension primer structure that is connected as a whole by at least the first extension primer and the second extension primer, and designs the binding site of the first extension primer on the template polynucleotide to be located upstream of the binding site of the second extension primer. The extension primer is added to the solid support of the single-stranded template polynucleotide of the array, and one situation is that the extension primer hybridizes and binds to one template polynucleotide chain; another situation is that two extension primers are hybridized and bound to one template polynucleotide chain, and each extension primer hybridizes and binds to two template polynucleotide chains.

[0016] In either case, in an environment with chain displacement activity, the extended chain of the first extension primer will displace the extended chain of the second extension primer during synthesis, and the extended chain formed by the second extension primer that is complementary to the template polynucleotide does not bind to the template polynucleotide chain, but is suspended in the sequencing environment to serve as the template chain for second-end sequencing; and because the first extension primer and the second extension primer are connected as a whole, the extended chain generated by the second extension primer is connected to the first extension primer through the second extension primer, and then bound to the template polynucleotide chain through the extended chain of the first extension primer, and then fixed on the solid support through the template polynucleotide chain, that is, by designing the first extension primer and the second extension primer connected as an integrated structure, the complementary chain of the template polynucleotide is generated by the principle of chain displacement amplification, and the complementary chain is indirectly fixed on the solid support, thereby improving the stability of the complementary chain in the cycle sequencing environment, improving the sequencing quality, and better solving the technical problems existing in the prior art.

[0017] In order to further improve the stability of the complementary chain, optionally, the extension primer also includes a third extension primer connected to the first extension primer and the second extension primer, and the binding site of the third extension primer on the template polynucleotide is the same as that of the first extension primer; in step S3, the extension primer hybridizes and binds to at least two parallel single-stranded template polynucleotides, and the third extension chain generated by the third extension primer and complementary to the template polynucleotide at least partially replaces the second extension chain generated by the second extension primer and complementary to the template polynucleotide.

[0018] Furthermore, the extension primer also includes a fourth extension primer connected to the first extension primer, the second extension primer, and the third extension primer, and the binding site of the third extension primer on the template polynucleotide is the same as that of the second extension primer; in step S3, the extension primer hybridizes and binds to at least two parallel single-stranded template polynucleotides, and the third extension chain generated by the third extension primer and complementary to the template polynucleotide at least partially replaces the fourth extension chain generated by the fourth extension primer and complementary to the template polynucleotide.

[0019] Regardless of whether the connecting structure of the extension primer is two, three or four, the principle of hybridization and binding reaction with the template polynucleotide chain is the same. It is only necessary to ensure that the template polynucleotide has spaced downstream binding sites and upstream binding sites, and different extension primers are connected as a whole, and a chain displacement amplification reaction occurs in an environment with chain displacement reaction activity.

[0020] The extension primers whose binding sites are located upstream are called upstream extension primers, and the extension primers whose binding sites are located downstream are called downstream extension primers. In order to further improve the orderliness and controllability of the strand displacement amplification reaction, the Tm value of the upstream extension primer in the extension primers is lower than the Tm value of the downstream extension primer; more preferably, the difference between the Tm values ​​of the upstream extension primer and the downstream extension primer is 5-10°C.

[0021] In order to further improve the controllability of the chain displacement amplification reaction, the extended chain generated by the extended primer at the downstream binding site in step S3 completely replaces the extended chain generated by the extended primer at the downstream binding site, and the displaced extended chain does not hybridize with the template polynucleotide and is completely overhanging.

[0022] In order to further improve the stability of the complementary chain, the 3' end of the extended chain generated by the extended primer of the upstream binding site also has an attachable portion, and the corresponding extended chain is fixed on the solid support through the attachable portion. Optionally, a ligand structure that matches the attachable portion of the extended chain is provided on the solid support; further optionally, the attachable portion of the extended chain is biotin, and the ligand structure on the corresponding solid support is streptavidin. Further optionally, the attachable portion of the extended chain is an azido group, an alkynyl group, a thiophosphate or a thiophosphate ester.

[0023] Optionally, the single-stranded template polynucleotide includes a first adaptor, a second adaptor, and a target DNA sequence inserted between the first adaptor and the second adaptor.

[0024] Optionally, the first adaptor includes a first sequencing primer binding site and an extension primer binding site; and the second sequencing primer is identical to at least part of the sequence of the second adaptor.

[0025] Optionally, the second adaptor further comprises an element for fixing the template polynucleotide on a solid support.

[0026] Optionally, the first adapter and the second adapter further include a tag sequence. The first adapter and the second adapter may use a universal adapter sequence, and by inserting different tag sequences into the adapter sequence, simultaneous detection of DNA sequences from different sources can be achieved.

[0027] Optionally, the first sequencing primer and the second sequencing primer have the same sequence.

[0028] Optionally, the sequences of the first sequencing primer and the second sequencing primer are different.

[0029] Optionally, the first extension primer and the second extension primer are connected as a whole through a linker structure, and the 5' ends of the first extension primer and the second extension primer are respectively modified to react chemically with the corresponding end structure of the linker structure to achieve covalent connection. Further optionally, the modified structure of the 5' ends of the first extension primer and the second extension primer includes an azide structure, and both ends of the linker are provided with an alkenyl or alkynyl structure that can undergo a click chemical reaction with the azide structure; further optionally, the linker structure also includes at least two functional groups, and the 5' ends of the third extension primer and the fourth extension primer respectively include a modified structure that can covalently bind to the corresponding functional group on the linker structure to achieve the connection of the first extension primer, the second extension primer, the third extension primer and the fourth extension primer as a whole; further optionally, the functional group of the linker structure can be an azide structure, and the modified structure of the 5' ends of the third extension primer and the fourth extension primer is an alkenyl or alkynyl structure that can react with the azide structure.

[0030] In order to maintain the flexibility of the connection structure of the first extension primer, the second extension primer, the third extension primer and the fourth extension primer, and avoid the connection structure from forming spatial restrictions on the hybridization and binding of each extension primer with the template chain, it is further preferred that the above-mentioned linker structure includes a 15-20 bp uracil chain; or the above-mentioned linker structure can also be a polymer molecular chain structure of 50-60 repeating units, for example, it can be a polyethylene glycol chain structure.

[0031] Optionally, a DNA polymerase having strand displacement activity is added to S3 to provide an environment for strand displacement reaction activity.

[0032] Furthermore, the DNA polymerase having strand displacement activity is phi29 polymerase, Bst polymerase, Bsu polymerase, Vent DNA polymerase, or Bsm DNA polymerase large fragment.

[0033] In order to improve the orderliness of the strand displacement amplification reaction and the quality of displacement amplification, the length of the spacer sequence between the upstream binding site and the downstream binding site of the extension primer on the template polynucleotide is 3 to 10 bp.

[0034] Optionally, the sequencing methods used in S1 and S4 include synthesis sequencing, pyrophosphate sequencing or ligation.

[0035] In addition, the present invention also provides a kit for paired-end sequencing of a template polynucleotide, comprising an extension primer for generating a sequencing polynucleotide chain complementary to the template polynucleotide; the extension primer comprises a first extension primer and a second extension primer connected as one, and the template polynucleotide has a binding site for the first extension primer and a binding site for the second extension primer; wherein the binding site of the first extension primer is located upstream of the binding site of the second extension primer.

[0036] Furthermore, the extension primers in the above kit also include a third extension primer connected to the first extension primer and the second extension primer as a whole, and the binding site of the third extension primer on the template polynucleotide is the same as that of the first extension primer.

[0037] Furthermore, the extension primers in the above kit also include a fourth extension primer connected to the first extension primer, the second extension primer, and the third extension primer, and the binding site of the fourth extension primer on the template polynucleotide is the same as that of the second extension primer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the principle of generating a second-end sequencing template strand in a specific embodiment of the present invention;

[0039] Figure 2 It is a schematic diagram of the principle of adding four extension primers connected as one body to hybridize with the template strand that completes the first end sequencing in a specific embodiment of the present invention;

[0040] Figure 3 This is a green fluorescence signal image of the sequencing method according to an embodiment of the present invention;

[0041] Figure 4 This is an image of a red fluorescent signal during the sequencing method according to an embodiment of the present invention. DETAILED DESCRIPTION

[0042] Below, in conjunction with specific embodiments, the present invention is further described, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] 1. Single-stranded template polynucleotide

[0044] The single-stranded template polynucleotide of the present invention refers to any polynucleotide chain in a single-stranded form, which serves as a template for first-end sequencing and may be a linear structure or a non-linear structure ("DNA nanoball"), preferably a single-stranded form in a linear structure;

[0045] The linear structure of the single-stranded template polynucleotide includes a first adapter, a second adapter, and a target DNA sequence inserted into the first adapter and the second adapter, the first adapter provides a first sequencing primer binding site and an extension primer binding site, and at least a portion of the sequence of the second adapter is identical to the second sequencing primer sequence; in some embodiments, the first adapter and the second adapter use universal sequences to achieve large-scale parallel sequencing of different target DNA sequences, and different target DNAs and sequencing directions can be marked by inserting tag sequences into the first adapter and the second adapter;

[0046] The single-stranded template polynucleotide of the non-linear structure (DNA nanoball) includes a single-stranded concatemer structure formed by at least two target DNA sequences. The first adapter and the second adapter are arranged at both ends of the single-stranded concatemer template polynucleotide. Different target DNA sequences are connected at intervals by a third adapter. The first adapter and the third adapter provide a first sequencing primer binding site and an extension primer binding site. At least part of the sequence of the third adapter and the second adapter is the same as the second sequencing primer sequence.

[0047] 2. Construction of single-stranded template polynucleotide library

[0048] 2.1 Overview of library construction methods

[0049] In many large-scale parallel sequencing technologies, there are many methods for generating sequencing template libraries. In certain embodiments, according to the library construction method commonly used in "Solexa" type sequencing, a linear structure single-stranded template polynucleotide library is constructed, the genomic DNA is first fragmented, and then both ends of the DNA fragments are connected to platform-specific oligonucleotide adapters, one end of the adapter is used to fix a single fragment on a solid support, and the single fragment is amplified in situ on the solid support to generate a single-stranded template polynucleotide cluster for sequencing;

[0050] In some other embodiments, a single-stranded template polynucleotide library with a non-linear structure can be constructed according to the DNA nanoball method developed by Drmanac et al., the genomic DNA can be fragmented, and single fragments can be used to produce circular DNA. Platform-specific oligonucleotide linkers can separate genomic DNA sequences (the separated genomic DNA sequences can be adjacent in the genome), and circular DNA can be amplified to produce single-stranded concatemers that can be fixed on a solid support.

[0051] It should be understood that the present invention adopts a library construction method known in the art to construct an array structure including many different target DNA sequences but sharing the same adapter sequence, thereby achieving large-scale parallel sequencing.

[0052] 2.2 Adaptors

[0053] The adaptors used in the construction of the above-mentioned single-stranded template polynucleotide library may include elements for fixing the template DNA polynucleotide on a solid support (e.g., the 5' end of the second adaptor carries a modification group that can react with a functional group on the solid support), and may also include a restriction endonuclease recognition site, an extension primer hybridization site, a barcode sequence, a unique molecular identifier sequence, and a polymerase recognition sequence (e.g., the extension primer binding site, sequencing primer binding site, and tag sequence provided on the first adaptor, the second adaptor, and the third adaptor in the above-mentioned template polynucleotide structure);

[0054] The adapter sequence can have a length, structure, and other properties suitable for a particular sequencing platform and intended use. For example, the adapter can be single-stranded, double-stranded, or partially double-stranded, and can be in the range of 10 to 200 nucleotides, 20 to 100 nucleotides, 40 to 100 nucleotides, or 50 to 80 nucleotides in length. In some embodiments, different members of a library will generally contain a common adapter sequence, although different species or subclasses in a library may have unique features, such as tag sequences or subgenus-specific barcodes;

[0055] A single adapter may include multiple subsequences with different functions. For example, the first adapter of the single-stranded template polynucleotide of the linear structure includes a first sequencing primer binding site and an extension primer binding site. Different functional sequences may overlap or not overlap according to actual needs, and may be arranged adjacently or at a certain distance. The overlapping region may overlap by 5%, 10%, 20%, 30%, 40% or 50%, and the non-overlapping may be separated by 1 to 10, 10 to 20, 30 to 40 or 40 to 50 nucleotides.

[0056] 2.3 Formation of single-stranded template polynucleotide clusters in an array structure fixed on a solid support

[0057] The present invention defines that the single-stranded template polynucleotide is fixed on the solid support in the form of an array, which can be fixed on a flat solid support surface in the form of an array, and all the single-stranded template polynucleotides in the array structure are in a completely identical and uniform reaction environment; or the solid support is provided with an array of recessed structures, and each single-stranded template polynucleotide is fixed in a corresponding recessed structure, and each recessed structure forms an independent reaction chamber;

[0058] In some embodiments, bridge amplification is used to form a single-stranded template polynucleotide in an array structure fixed on a solid support;

[0059] In some embodiments, a non-bridge amplification method is used, and a linker sequence that can hybridize and bind to a linker at one end of the fragmented DNA (usually used as the second linker of the formed single-stranded template polynucleotide) is pre-fixed on the solid support, and a clustering method similar to the amplification method disclosed by Zhaochun Ma et al. in the paper "Isothermal amplification method for next-generation sequencing" is used to form a single-stranded template polynucleotide array structure fixed on the solid support; it should be understood that the present invention can also use other methods that can amplify single-stranded fragmented DNA to form an array by pre-fixing a linker on a solid support. The cluster formation method does not determine the implementation of the sequencing method of the present invention;

[0060] In some embodiments, a nucleotide template having a single-stranded polymer structure can be formed using the DNA nanoball method developed by Drmanac et al. and immobilized in the recessed structure of the array.

[0061] 3. Sequencing of the first end of single-stranded template polynucleotide

[0062] In some embodiments, the first end sequencing method of the single-stranded template polynucleotide includes providing a single-stranded template polynucleotide cluster in an array fixed on a solid support according to the above method, setting a first adaptor at the 3' end of the single-stranded template polynucleotide, and fixing the 5' end on the solid support through a second adaptor, a first sequencing primer binding site is set on the first adaptor, adding a first sequencing primer, and the first sequencing primer binds to a corresponding position of the first adaptor, and the first end sequencing of the single-stranded template polynucleotide can be performed by a synthetic sequencing method, a ligation sequencing method, or a pyrophosphate sequencing method to obtain an extended polynucleotide chain of the first sequencing primer and the corresponding first end sequence information;

[0063] 4. Generation of complementary strands of single-stranded template polynucleotides and second-end sequencing

[0064] The principle of generating complementary strands of single-stranded template polynucleotides is as follows Figure 1 As shown, the extended polynucleotide chain of the first sequencing primer is removed, and an extension primer is added; wherein the extension primer includes a first extension primer and a second extension primer connected as a whole, and the template polynucleotide has a binding site for the first extension primer and a binding site for the second extension primer; wherein the binding site of the first extension primer is located upstream of the binding site of the second extension primer;

[0065] The extension primer is annealed and hybridized with the template polynucleotide, the first extension primer binding site and the second extension primer binding site of the template polynucleotide are correspondingly bound to the first extension primer and the second extension primer, in an environment with chain displacement reaction activity, the first extension chain complementary to the template polynucleotide generated by the first extension primer at least partially replaces the second extension chain complementary to the template polynucleotide generated by the second extension primer, and there is at least a portion of the second extension chain that is not hybridized and bound to the template polynucleotide, which is called the overhang portion of the second extension chain. The overhang portion of the second extension chain is the complementary chain of the template polynucleotide, and serves as the template chain for sequencing the second end of the single-stranded template polynucleotide.

[0066] In some embodiments, in order to further improve the controllability of the chain displacement amplification reaction, the extended chain generated by the extension primer of the downstream binding site in step S3 completely replaces the extended chain generated by the extension primer of the downstream binding site, and the displaced extended chain does not hybridize and bind to the template polynucleotide and is completely overhanging. Since the extension primer of the downstream binding site of the present invention can be indirectly fixed on the solid support through the upstream extension primer, the extended chain of the upstream extension primer and the template polynucleotide chain in sequence, even if the extended chain of the displaced downstream extension primer is completely overhanging, it can still be stably fixed on the solid support. In the process of the chain displacement reaction, there is no need to control the progress of the chain displacement by reaction conditions such as temperature, enzyme activity, reaction time, etc., thereby improving the controllability of the entire sequencing process and solving the technical problems not solved in the prior art.

[0067] In some embodiments, the extension primer whose binding site is located upstream is called an upstream extension primer, and the extension primer whose binding site is located downstream is called a downstream extension primer. In order to improve the orderliness of the chain displacement amplification reaction and the quality of displacement amplification, the Tm value of the upstream extension primer is lower than the Tm value of the downstream extension primer; further preferably, in some embodiments, the difference in Tm values ​​between the upstream extension primer and the downstream extension primer is 5~10°C; further preferably, in some embodiments, the length of the spacer sequence between the upstream primer binding site and the downstream primer binding site is 3~10bp.

[0068] The overall scheme for achieving paired-end sequencing is usually to first complete at least partial sequencing of the single-stranded template polynucleotide to obtain the sequence information of the first end; then generate the complementary chain of the single-stranded template polynucleotide for sequencing to obtain the sequence information of the second end. However, the most important problem in the implementation of this overall scheme is how the generated complementary chain can be stably present in the environment of cyclic sequencing and flushing, that is, the most important problem is how to stably fix the generated complementary chain on the solid support. In order to solve the above problems, the present invention specially designs an extension primer for complementary hybridization extension with the template polynucleotide to generate a complementary chain, forming an extension primer structure that is connected as a whole by at least the first extension primer and the second extension primer, and designs the binding site of the first extension primer on the template polynucleotide to be located downstream of the second extension primer binding site. The extension primer is added to the solid support of the single-stranded template polynucleotide of the array, and one situation is that the extension primer hybridizes and binds to one template polynucleotide chain; another situation is that two extension primers are hybridized and bound to one template polynucleotide chain, and each extension primer hybridizes and binds to two template polynucleotide chains.

[0069] In either case, in an environment with chain displacement activity, the extended chain of the first extension primer will displace the extended chain of the second extension primer during synthesis, and the extended chain formed by the second extension primer that is complementary to the template polynucleotide does not bind to the template polynucleotide chain, but is suspended in the sequencing environment and serves as the template chain for second-end sequencing; and because the first extension primer and the second extension primer are connected as a whole, even if the extended chain generated by the second extension primer is completely displaced and completely separated from the template polynucleotide chain, the extended chain generated by the second extension primer can still be connected to the first extension primer through the second extension primer, and then combined with the template polynucleotide chain through the extended chain of the first extension primer, and then fixed on the solid support through the template polynucleotide chain, that is, by designing the first extension primer and the second extension primer connected as an integrated structure, the complementary chain of the template polynucleotide is generated by the principle of chain displacement amplification, and the complementary chain is indirectly fixed on the solid support, thereby improving the stability of the complementary chain in the cycle sequencing environment, improving the sequencing quality, and better solving the technical problems existing in the prior art.

[0070] In order to further improve the stability of the complementary chain, in some embodiments, the extension primer also includes a third extension primer connected to the first extension primer and the second extension primer, and the binding site of the third extension primer on the template polynucleotide is the same as that of the first extension primer; in step S3, the extension primer hybridizes and binds to at least two parallel single-stranded template polynucleotides, and the third extension chain generated by the third extension primer and complementary to the template polynucleotide at least partially replaces the second extension chain generated by the second extension primer and complementary to the template polynucleotide.

[0071] Furthermore, in some embodiments, the extension primer further comprises a fourth extension primer connected to the first extension primer, the second extension primer, and the third extension primer, and the binding site of the third extension primer on the template polynucleotide is the same as that of the second extension primer; in step S3, the extension primer hybridizes with at least two parallel single-stranded template polynucleotides, such as Figure 2 As shown, the third extended strand generated by the third extension primer and complementary to the template polynucleotide at least partially replaces the fourth extended strand generated by the fourth extension primer and complementary to the template polynucleotide.

[0072] It should be understood that no matter whether the connecting composition structure of the extension primer is two, three or four, the principle of hybridization binding reaction with the template polynucleotide chain is the same. It is only necessary to ensure that the template polynucleotide has spaced downstream binding sites and upstream binding sites, and different extension primers are connected as a whole, and a chain displacement amplification reaction occurs in an environment with chain displacement reaction activity.

[0073] In addition, in some embodiments, in order to further improve the stability of the complementary chain, the 3' end of the extended chain generated by the extended primer of the upstream binding site also has an attachable portion, and the corresponding extended chain is fixed on the solid support through the attachable portion. Optionally, a ligand structure that matches the attachable portion of the extended chain is provided on the solid support; further optionally, the attachable portion of the extended chain is biotin, and the ligand structure on the corresponding solid support is streptavidin.

[0074] 5. Ligation of extension primers

[0075] The first extension primer and the second extension primer are connected as a whole through a linker structure, and the 5' ends of the first extension primer and the second extension primer are respectively modified to chemically react with the corresponding end structure of the linker structure to achieve covalent connection.

[0076] In some embodiments, the 5'-end modification structures of the first extension primer and the second extension primer include an azide structure, and both ends of the linker are provided with an alkene or alkynyl structure capable of undergoing a click chemistry reaction with the azide structure;

[0077] Furthermore, in some embodiments, the linker structure also includes at least two functional groups, and the 5' ends of the third extension primer and the fourth extension primer respectively include modified structures that can be covalently bonded to the corresponding functional groups on the linker structure to achieve the connection of the first extension primer, the second extension primer, the third extension primer and the fourth extension primer as one; further optionally, the functional group of the linker structure can be an azide structure, and the modified structure at the 5' end of the third extension primer and the fourth extension primer is an alkene or alkynyl structure that can react with the azide structure.

[0078] In order to maintain the flexibility of the connection structure of the first extension primer, the second extension primer, the third extension primer and the fourth extension primer and avoid the connection structure from forming spatial restrictions on the hybridization and binding of each extension primer with the template chain, in some embodiments, the linker structure includes a 15-20 bp uracil chain.

[0079] 6. Formation of a strand displacement active reaction environment

[0080] Strand displacement activity refers to the phenomenon that biological, chemical or physical agents cause paired nucleic acids to dissociate from their complementary chains in the 5' to 3' direction, bind and approach template-dependent nucleic acid synthesis, and displace downstream DNA encountered during the synthesis process; strand displacement begins at the 5' end of the paired nucleic acid sequence, so the enzyme immediately performs nucleic acid synthesis at the 5' end of the displacement site. The newly synthesized nucleic acid and the displaced nucleic acid usually have the same nucleotide sequence complementary to the template nucleic acid chain. The strand displacement activity can be located on the same molecule as the molecule that confers the activity of nucleic acid synthesis, or it can be a separate and independent activity. For example, DNA polymerases, such as Escherichia coli DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase of T7 or T5 phages, and HIV viral transcriptase, are enzymes with both polymerase activity and strand displacement activity. Reagents such as helicases can be used in combination with inducers that do not have strand displacement activity to produce a strand displacement effect.

[0081] In some embodiments of the present invention, the strand displacement activity reaction environment is provided by using a DNA polymerase having strand displacement activity, such as phi29 polymerase, Bst polymerase, Bsu DNA polymerase, Vent DNA polymerase, and BsmDNA polymerase large fragment.

[0082] 7. Second end sequencing of single-stranded template polynucleotide

[0083] In an embodiment of the present invention, a second adapter of a known sequence is set at the 3' end of the template polynucleotide sequenced at the first end. During the amplification and extension process of the extension primer using the corresponding single-stranded template polynucleotide as a template, the extended chain of the upstream binding site extension primer (the extended chain of the first extension primer) replaces the extended chain of the downstream binding site extension primer (the extended chain of the second extension primer), and the extended chain of the downstream binding site extension primer is complementary to the corresponding template polynucleotide, including forming a sequence complementary to the second adapter of the template polynucleotide at the portion near the 5' end. During the process of the upstream extended chain replacing the downstream extended chain, the sequence complementary to the second adapter at the 5' end is at least partially replaced by an overhang, and in some embodiments, the overhang is completely replaced. When a second sequencing primer having at least a partial sequence identical to the second adapter is added, the second sequencing primer hybridizes and binds to at least a portion of the sequence complementary to the second adapter at the overhang portion of the downstream extended chain near the 5' end, and the second end sequencing of the single-stranded template polynucleotide is performed using the extended chain of the downstream binding site extension primer as a template by a synthetic sequencing method, a ligation sequencing method, or a pyrophosphate sequencing method. Example

[0084] 1. Construction of single-stranded nucleic acid template:

[0085] The first adapter sequence was designed as:

[0086] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; the second adapter sequence is:

[0087] 5'-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG-3'; the phix174 genome sequence with known sequence was used as the nucleotide chain to be sequenced.

[0088] The first adapter and the second adapter are connected to the two ends of the nucleotide chain to be sequenced, and the amplification clustering method disclosed by Zhaochun Ma et al. in the paper "Isothermal amplification method for next-generation sequencing" is used to form a single-stranded template polynucleotide with an array structure fixed on the chip;

[0089] 2. First-end sequencing:

[0090] The first sequencing primer was designed to be 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; 1uM of the first sequencing primer was added to the flow channel of the chip reaction chamber where the single-stranded template polynucleotide cluster was formed, and reversible terminator nucleotides labeled with different fluorescent dyes were used as modified nucleotide raw materials, and sequencing was performed at 60°C for 50 cycles; then, the double-stranded structure was denatured and the first sequencing primer extension chain was washed away;

[0091] 3. Second end sequencing:

[0092] Design the extension primer structure as

[0093] '

[0094] Add 2 μM of extension primer to the flow channel of the chip reaction chamber that completes the first-end sequencing, and extend the extension primer in a mixture containing Phi29 polymerase and dNTP at 50° C. for 30 minutes to form a complementary chain of the single-stranded template polynucleotide that completes the first-end sequencing;

[0095] The second sequencing primer was designed to be 5'-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3'; 1 μM of the second sequencing primer was added to the flow channel of the chip reaction chamber, and reversible terminator nucleotides labeled with different fluorescent dyes were used as modified nucleotide raw materials, and sequencing was performed at 60°C for 50 cycles;

[0096] 4. Determination of sequence

[0097] The fluorescence signal image information obtained by the first-end sequencing and the second-end sequencing is processed and integrated, such as Figure 3 and Figure 4 As shown in Table 1, the results show that the nucleotide chain to be sequenced is sequenced correctly.

[0098] Table 1

[0099] library Sequencing cycle number Density (k / mm2) Cluster PF (%) phas / prephas %>=Q30 Error rate phix174 PE50 236 79.3 Read1:0.230 / 0.166 Read2:0.238 / 0.191 Read1:87.4Read2:88.6 Read1: 0.91 Read2: 0.78

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A template polynucleotide paired-end sequencing method, characterized in that: The steps include: S1. providing a single-stranded template polynucleotide cluster in an array fixed on a solid support, adding a first sequencing primer, sequencing at least a portion of the sequence of the single-stranded template polynucleotide, and obtaining an extended polynucleotide chain of the first sequencing primer and corresponding first terminal sequence information; S2, removing the extended polynucleotide chain of the first sequencing primer and adding an extension primer; The extension primer includes a first extension primer and a second extension primer connected as a whole through a linker structure at the 5' end, and the template polynucleotide has a binding site for the first extension primer and a binding site for the second extension primer; wherein the binding site of the first extension primer is located upstream of the binding site of the second extension primer; S3, annealing and hybridizing the extension primer and the template polynucleotide, in an environment with strand displacement reaction activity, the extension chain generated by the first extension primer completely replaces the extension chain generated by the second extension primer, and the displaced extension chain does not hybridize and bind to the template polynucleotide and is completely overhanging; S4, adding a second sequencing primer to sequence the overhang portion of the second extended chain to obtain the corresponding second terminal sequence information, thereby completing paired-end sequencing of the template polynucleotide; The Tm value of the first extension primer is lower than the Tm value of the second extension primer, and the difference between the Tm values ​​of the first extension primer and the second extension primer is 5-10°C.

2. The method for sequencing paired ends of a template polynucleotide according to claim 1, wherein: The single-stranded template polynucleotide includes a first adaptor, a second adaptor, and a target DNA sequence inserted between the first adaptor and the second adaptor.

3. The method for sequencing paired ends of a template polynucleotide according to claim 2, wherein: The first adaptor comprises a first sequencing primer binding site and an extension primer binding site; the second sequencing primer has at least a partial sequence identical to that of the second adaptor.

4. The method for sequencing paired ends of a template polynucleotide according to claim 3, wherein: The second adaptor also includes an element for fixing the template polynucleotide to a solid support.

5. The method for sequencing paired ends of a template polynucleotide according to claim 4, characterized in that: The first adaptor and the second adaptor further include a tag sequence.

6. The method for sequencing paired ends of a template polynucleotide according to any one of claims 1 or 2, characterized in that: The first sequencing primer and the second sequencing primer have different sequences.

7. The method for sequencing paired ends of a template polynucleotide according to any one of claims 1 or 2, characterized in that: The first extension primer and the second extension primer connected as a whole through the linker structure at the 5' end are specifically the first extension primer and the second extension primer whose 5' ends are respectively modified to chemically react with the corresponding end structure of the linker structure to achieve covalent connection.

8. The method for sequencing paired ends of a template polynucleotide according to claim 7, wherein: The linker structure includes a 15-20bp uracil chain or a polymer molecule chain of 50-60 repeating units.

9. The method for sequencing paired ends of a template polynucleotide according to any one of claims 1 or 2, characterized in that: The 3' end of the extended chain generated by the first extended primer also has an adaptable portion, and the corresponding extended chain is fixed on the solid support through the adaptable portion.

10. The method for sequencing paired ends of a template polynucleotide according to claim 9, wherein: Such attachable moieties include alkynyl, azide, phosphorothioate, phosphorothioate, or biotin.

11. The method for sequencing paired ends of a template polynucleotide according to any one of claims 1 or 2, characterized in that: S3 provides an environment for strand displacement reaction activity by adding a DNA polymerase having strand displacement activity.

12. The method for sequencing paired ends of a template polynucleotide according to any one of claims 11, characterized in that: The DNA polymerase with strand displacement activity is phi29 polymerase, Bst polymerase, Bsu DNA polymerase, Vent DNA polymerase, and Bsm DNA polymerase large fragment.

13. The method for sequencing a template polynucleoside paired end according to any one of claims 1 or 2, characterized in that: The length of the spacer sequence between the binding site of the first extension primer and the binding site of the second extension primer is 3 to 10 bp.

14. The method for sequencing paired ends of a template polynucleotide according to any one of claims 1 or 2, characterized in that: The sequencing methods used in S1 and S4 include synthesis sequencing, pyrosequencing or ligation.

15. A kit for paired-end sequencing of a template polynucleotide, characterized in that: It includes an extension primer for generating a sequencing polynucleotide chain complementary to a template polynucleotide; the extension primer includes a first extension primer and a second extension primer connected as a whole by a linker structure at the 5' end, and the template polynucleotide has a binding site for the first extension primer and a binding site for the second extension primer; wherein the binding site of the first extension primer is located upstream of the binding site of the second extension primer; In an environment with strand displacement activity, the extended strand generated by the first extension primer completely displaces the extended strand generated by the second extension primer, and the displaced extended strand does not hybridize and bind to the template polynucleotide and is completely overhanging; The Tm value of the first extension primer is lower than the Tm value of the second extension primer, and the difference between the Tm values ​​of the first extension primer and the second extension primer is 5-10°C.

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