Paired-end sequencing methods and kits

Abstract

A double-end sequencing method of the present invention, a double-stranded template polynucleotide to be tested is loaded into a sequencing chip with primers fixed on the surface for amplification, and the first template chain to be tested is subjected to SBS sequencing; then the second template chain to be tested is obtained by extension synthesis; a dNTP modified with a second active group is connected to the end of the second template chain to be tested, and then the second active group is subjected to a click reaction with the first active group located on the surface of the sequencing chip, and the second template chain to be tested is subjected to SBS sequencing. The double-end sequencing method of the present invention directly amplifies and extends the second template chain to be tested after sequencing the first template chain to be tested, and the second template chain to be tested is fixed to the solid surface by chemical bonds for sequencing, thereby simplifying the process of generating the second template chain to be tested fixed to the solid surface, reducing damage to the chip substrate and DNA, and reducing the cost of amplification sequencing.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid sequencing, and in particular relates to a double-end sequencing method and a kit. Background Art

[0002] In recent years, nucleic acid sequencing technology has developed rapidly, mainly including first-generation sequencing technology, second-generation sequencing technology and third-generation sequencing technology, and the fourth-generation sequencing technology has also developed rapidly in recent years. Nucleic acid sequencing technology has gradually become more high-throughput, low-cost and multifunctional. However, the second-generation sequencing technology still occupies a major position in the global sequencing market. The second-generation sequencing technology is also called the next-generation sequencing technology NGS, which is based on massive parallel sequencing technology (MPS) and is generally referred to as high-throughput sequencing technology in the industry. It can simultaneously complete the synthesis of complementary chains of sequencing templates and the acquisition of sequence information. Compared with the first-generation sequencing technology, it has lower costs, more tests at one time, and shorter sequencing cycles.

[0003] At present, the commonly used polynucleotide sequencing methods in the second-generation high-throughput sequencing include sequencing by synthesis (SBS); it mainly combines the principles and technologies of DNA hybridization and polymerase chain reaction, and obtains the test template by making multiple copies of the test polynucleotide involving primers. There are specific primer hybridization sites on the test template chain. After the test template is processed, it is hybridized with a specific primer. After adding fluorescently labeled nucleotides, the 3' end of the specific primer is extended with a nucleotide complementary to the template chain under the action of polymerase. The 3' end of the nucleotide can also be modified with a specific blocking group so that only one nucleotide can be extended at a time. After determining the type of the extended nucleotide, the 3' end blocking group and the fluorescent labeling group are removed, so that it can be reactivated and the next nucleotide can be extended. This cycle can realize the reading of the sequence of the test chain template chain.

[0004] Paired-end sequencing is one of the important technologies for high-throughput sequencing. It is a technology for sequencing the positive strand and the reverse complementary strand of the nucleic acid strand to be tested, such as patents US10457985B2, WO2008041002, US20060292611, W006135342, W003074734, US20060024681, US7754429B2, etc., which all describe related double-end sequencing methods. They are all achieved by means of solid surface amplification methods, such as bridge amplification, which is a solid surface amplification method. This method is to fix two primers on the solid surface, and the polynucleotide to be tested is amplified by the primers fixed on the surface to obtain a group or cluster of polynucleotide molecules to be tested. The cluster is ultimately covalently fixed to the solid surface and can be used as a sequencing template for SBS sequencing.

[0005] Patent CN101663405A describes several methods for achieving double-end sequencing, wherein two primers with different restriction sites are fixed on the surface of a solid support, and a first template chain and a second template chain are obtained on the solid support through bridge amplification, wherein the first template chain and the second template chain are both fixed to the surface through the 5' end, and the second template chain is selectively removed to leave the first template chain, and the first template chain can hybridize with the primer fixed on the solid surface. After sequencing the first template chain, the primer that has been removed from the surface needs to be repaired to have extension activity again, and then the second template chain is re-amplified by the repaired surface primer to regain the second template chain; after selectively removing the first template chain, the second template chain can be sequenced. However, the above-mentioned double-end sequencing method has the following disadvantages: the process of generating the second template chain is complicated, which not only requires the primers that have been excised on the surface to be repaired to have extension activity again, but also requires multiple cycles of amplification to construct the second template chain for sequencing; the denaturing reagents used in the amplification cycle are generally highly destructive reagents such as alkali or formamide. The repeated use of such reagents not only affects the sequencing chip substrate and causes the surface material to fall off, but also damages the DNA, affecting the accuracy of the sequencing results. Summary of the invention

[0006] Based on this, the present invention provides a double-end sequencing method, which can simplify the sequencing process, ensure the accuracy of the sequencing results, reduce the use of highly destructive reagents in the amplification process, and reduce the cost of amplification sequencing.

[0007] The first aspect of the present invention provides a double-end sequencing method, comprising the following steps:

[0008] S1, loading a double-stranded template polynucleotide to be tested on a sequencing chip with fixed primers fixed on the surface for amplification, to obtain a double-stranded polynucleotide to be tested fixed on the surface of the sequencing chip; the surface of the sequencing chip is modified with a first active group, and the fixed primers include a P5 primer and a P7 primer having different restriction sites;

[0009] S2, selectively removing the second test template strand extended by the P5 primer in the test polynucleotide double strand, leaving the first test template strand extended by the P7 primer, and then performing a first sequencing on the first test template strand;

[0010] S3, using the first template strand to be tested as a template, replicating and synthesizing a second template strand to be tested that is complementary to the first template strand to be tested;

[0011] S4, connecting the end of the second test template chain synthesized in step S3 with a dNTP modified with a second active group; performing a click reaction between the second active group and the first active group to fix the second test template chain on the surface of the sequencing chip; selectively removing the first test template chain, and then performing a second sequencing on the second test template chain.

[0012] In some embodiments, in step S2, the first sequencing includes: performing a first SBS sequencing on the first template strand to be tested using the SP1 primer to obtain a sequencing strand;

[0013] Step S3 includes: using the first test template strand as a template, extending and synthesizing an extended strand on the sequencing strand to obtain a second test template strand, wherein the second test template strand is sequentially composed of an SP1 primer, a sequencing strand and an extended strand.

[0014] The second aspect of the present invention provides a double-end sequencing kit, comprising:

[0015] A sequencing chip, the surface of which is modified with a first active group, and the surface of the sequencing chip has different restriction sites, a P5 primer and a P7 primer, fixed thereon;

[0016] The terminal modification reagent includes TDT terminal transferase and dNTP modified with a second active group, wherein the TDT terminal transferase is used for transferring the dNTP modified with the second active group, and the second active group is used for generating a click reaction with the first active group to form a chemical chain.

[0017] In the double-end sequencing method in the prior art, after sequencing the first template chain, the primers that have been excised on the surface need to be repaired to have extension activity again, and then the second template chain fixed on the solid surface is obtained again by amplification through the repaired surface primers, so as to sequence the first template chain. In order to overcome the problem of cumbersome processes such as repairing the joint and amplification cycles required in generating the second template chain fixed on the solid surface; a double-end sequencing method of the present invention fixes the first template chain to be tested on the solid surface for sequencing, replicates and synthesizes the second template chain to be tested that is not fixed on the solid surface, and then connects the end of the second template chain to be tested with a dNTP modified with a second active group, and forms a link through a click reaction between the second active group and the first active group modified on the surface of the sequencing chip, thereby fixing the second template chain to be tested on the surface of the sequencing chip for sequencing. The present invention proposes a sequencing method that first replicates and synthesizes a second test template chain that is not fixed on a solid phase surface, and then performs a fixed sequencing of the second test template chain. This simplifies the process of generating the second test template chain fixed on a solid phase surface by means of amplification cycles, eliminates the step of repairing the joint, simplifies the sequencing process, and reduces the use of highly destructive reagents in the amplification process. At the same time, it can reduce the cost of amplification sequencing and ensure the accuracy of the sequencing results.

[0018] Furthermore, the present invention continues to perform extension synthesis on the sequencing chain formed after the sequencing of the first template chain to be tested is completed, so as to obtain a second template chain to be tested that is complementary to the first template chain to be tested, that is, the sequencing chain is fully utilized, and the use of strong destructive reagents to remove the sequencing chain to damage the DNA, thereby affecting the accuracy of the sequencing result, and the length of the extended chain further extended and synthesized on the sequencing chain is reduced, thereby improving the synthesis efficiency of the second template chain to be tested.

[0019] Furthermore, the present invention utilizes chemical bonds to fix the second template chain to be tested on the solid surface, and preferably a linking bond is formed by a click reaction. The reaction conditions are simple and easy to control, and the reaction has strong stereoselectivity, rapid reaction, high reaction efficiency, harmless by-products, and the products are insensitive to oxygen and water and will not damage DNA. It is an ideal way to fix the second template chain to be tested by chemical bonds.

[0020] The double-end sequencing kit of the present invention has a simple sequencing process, does not need to repair the surface joint, and does not need to perform an amplification cycle in the generation of the second template chain to be tested, reduces the use of denaturing reagents in the amplification process, thereby reducing damage to the chip substrate and DNA, and has high sequencing accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a schematic diagram of the synthesis of a first template strand to be tested and a second template strand to be tested in a double-end sequencing method.

[0022] Figure 2 It is a schematic diagram of sequencing the second template strand to be tested in a double-end sequencing method of the present invention.

[0023] Figure 3 This is the original signal diagram of the same round of sequencing of the first test template strand and the second test template strand in Example 1 of the present invention; wherein, Figure 3 A is the first template chain to be tested, Figure 3 B is the second template chain to be tested.

[0024] Figure 4 It is a basecall analysis diagram of the sequencing signals of the first test template chain and the second test template chain in Example 1 of the present invention; wherein, cycle1 to cycle75 are 75bp sequencing signals of the first test template chain, and cycle76 to cycle150 are 75bp sequencing signals of the second test template chain; lines A, C, G and T correspond to the fluorescence signals of the A, C, G and T bases respectively.

[0025] Figure 5 This is a distribution curve of base mass scores of each round of the first template strand to be tested and the second template strand to be tested in Example 1 of the present invention.

[0026] Figure 6 This is the original signal diagram of the same round of sequencing of the first test template strand and the second test template strand in Example 2 of the present invention; wherein, Figure 3 A is the first template chain to be tested, Figure 3 B is the second template chain to be tested.

[0027] Figure 7 It is a basecall analysis diagram of the sequencing signals of the first test template chain and the second test template chain in Example 2 of the present invention; wherein, cycle1 to cycle75 are 75bp sequencing signals of the first test template chain, and cycle76 to cycle150 are 75bp sequencing signals of the second test template chain; lines A, C, G and T correspond to the fluorescence signals of the A, C, G and T bases respectively.

[0028] Figure 8 This is a distribution curve of base mass scores in each round of the first template strand to be tested and the second template strand to be tested in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] A double-end sequencing method comprises the following steps:

[0032] S1, loading a double-stranded template polynucleotide to be tested on a sequencing chip with fixed primers fixed on the surface for amplification, to obtain a double-stranded polynucleotide to be tested fixed on the surface of the sequencing chip; the surface of the sequencing chip is modified with a first active group, and the fixed primers include a P5 primer and a P7 primer having different restriction sites;

[0033] S2, selectively removing the second test template strand extended by the P5 primer in the test polynucleotide double strand, leaving the first test template strand extended by the P7 primer, and then performing a first sequencing on the first test template strand;

[0034] S3, using the first template strand to be tested as a template, replicating and synthesizing a second template strand to be tested that is complementary to the first template strand to be tested;

[0035] S4, connecting the end of the second test template chain synthesized in step S3 with a dNTP modified with a second active group; performing a click reaction between the second active group and the first active group to fix the second test template chain on the surface of the sequencing chip; selectively removing the first test template chain, and then performing a second sequencing on the second test template chain.

[0036] The amplification method in step S1 includes but is not limited to: one or a combination of two or more amplification methods such as bridge amplification, loop-mediated isothermal amplification, recombinase polymerase amplification, template walking amplification, etc. Preferably, the amplification method in the following embodiments is bridge amplification.

[0037] In some embodiments, in step S2, the first sequencing includes: performing a first SBS sequencing on the first template strand to be tested using the SP1 primer to obtain a sequencing strand;

[0038] Step S3 includes: using the first test template strand as a template, extending and synthesizing an extended strand on the sequencing strand to obtain a second test template strand, wherein the second test template strand is sequentially composed of an SP1 primer, a sequencing strand and an extended strand.

[0039] In some embodiments, the fixed primer is covalently fixed to the surface of the sequencing chip via the 5' end; and / or,

[0040] The 5' end to 3' end of the second test template strand synthesized in step S3 are sequentially composed of SP1 primer, sequencing strand and extension strand, and the second test template strand is fixed to the surface of the sequencing chip through the 3' end.

[0041] In some embodiments, the selective removal of the second test template chain comprises: performing enzyme cleavage on the restriction site of the P5 primer, and then denaturing and eluting to remove the second test template chain complementary to the first test template chain; and / or,

[0042] The selective excision of the first test template chain includes: performing enzyme cleavage on the enzyme cleavage site of the P7 primer, and then denaturing and eluting to remove the first test template chain that is complementary hybridized with the second test template chain.

[0043] In some embodiments, in step S4, connecting the dNTP modified with the second active group to the end of the second template chain to be tested includes: adding terminal transferase and the dNTP modified with the second active group to the second template chain to be tested synthesized in step S3, so that the end of the second template chain to be tested is connected to the dNTP modified with the second active group.

[0044] In some embodiments, the click reaction comprises an azide-alkyne cycloaddition reaction or a reverse electron demand Diels-Alder coupling reaction.

[0045] In some embodiments, the structural formula of the dNTP modified with the second reactive group is shown in Formula I;

[0046]

[0047] Wherein, the R1 is selected from azido, 1-methylcyclopropenyl, C2-C4 alkynyl or C2-C4 alkenyl, and Base is a base.

[0048] In some embodiments, the structural formula of the dNTP modified with the second active group is shown in Formula II;

[0049]

[0050] Wherein, R2 is selected from 1,2,4-triazine, 1-methylcyclopropene, trans-cyclooctenyl, azido, C2-C4 alkynyl or C2-C4 alkenyl; R3 is selected from H, C1-C3 alkyl, and Base is a base.

[0051] In some embodiments, the first reactive group is selected from an azido group, a C2-C4 alkynyl group, a trans-cyclooctenyl group, a 1,2,4,5-tetrazine group or a 1-methylcyclopropene group.

[0052] In some embodiments, the P5 includes a restriction site ideoxyU; and / or, the P7 includes a restriction site i8oxodG.

[0053] In some of the embodiments, the base sequence of P5 is shown as SEQ ID NO: 1; and / or, the base sequence of P7 is shown as SEQ ID NO: 2.

[0054] In some embodiments, the base sequence of the SP1 primer is shown in SEQ ID NO: 3; and / or,

[0055] The base sequence of the SP2 primer used for SBS sequencing of the second template strand to be tested is shown in SEQ ID NO:4.

[0056] A double-end sequencing method of the present invention also includes another embodiment:

[0057] In step S2, the first test template chain extended by the P7 primer in the double-stranded polynucleotide to be tested is selectively excised, leaving the second test template chain extended by the P5 primer, and then the second test template chain is sequenced for the first time; similarly, in the subsequent steps, the processing order of the first test template chain and the second test template chain in the above-mentioned double-end sequencing method is replaced, thereby achieving sequencing of the second test template chain and the first test template chain in sequence.

[0058] A double-end sequencing kit, comprising:

[0059] A sequencing chip, the surface of which is modified with a first active group, and the surface of the sequencing chip has different restriction sites, a P5 primer and a P7 primer, fixed thereon;

[0060] The terminal modification reagent includes TDT terminal transferase and dNTP modified with a second active group, wherein the TDT terminal transferase is used for transferring the dNTP modified with the second active group, and the second active group is used for generating a click reaction with the first active group to form a chemical chain. The following are specific embodiments.

[0061] Example 1

[0062] 1. Preparation of sequencing chip with modified P5 and P7 primers

[0063] 1) obtaining a sequencing chip chemically modified with an azide group;

[0064] 2) Design and synthesize P5 and P7 primers, use 3×SSC solution to prepare primer solutions with a concentration of 5uM each, and pass them into the chip flow channel, and heat them in a 55°C oven for 4 hours. The 5' ends of the two primers are modified with DBCO groups, and the primers are fixed to the chip surface through the reaction of the DBCO groups at their 5' ends with the azide groups on the chip surface. Among them,

[0065] The base sequence of P5 primer (SEQ ID NO: 1):

[0066] 5'-TTTTTTTTAA / ideoxyU / GATACGGCGACCACCGAGATCTACAC-3'

[0067] The base sequence of P7 primer (SEQ ID NO: 2):

[0068] 5'-TTTTTTTTCAAGCAGAA / i8oxodG / ACGGCATACGAGAT-3'.

[0069] 3) After the reaction is completed, each flow channel is cleaned with 3×SSC solution and set aside; the obtained sequencing chip can be used for subsequent amplification sequencing.

[0070] 2. Amplification of the library to be tested on the sequencing chip and first-strand sequencing

[0071] 2.1 Experimental Materials

[0072] The library to be tested was constructed from Escherichia coli ATCC8739, and the length of the library fragment was 450 bp. The library was obtained by purchasing a commercially available library construction kit (Vahts Universal DNA Library Prep Kit for Illumina V4, #ND610-1).

[0073] After denaturing the double-stranded library to be tested with 0.1 M NaOH solution, 3×SSC buffer was used to prepare a 2 pM loading solution.

[0074] 2.2, amplification and sequencing, such as Figure 1 As shown, the steps include:

[0075] 1) Template hybridization

[0076] Load the sequencing chip and the denatured 2pM library loading solution into a device with temperature control and microfluidic modules for amplification, such as the Salus Pro system (matching kit). Each reagent in the reaction can be pumped into the chip through the microfluidic module of the Salus Pro system and the corresponding temperature can be set through the heating module to react;

[0077] After the library (double-stranded template polynucleotide to be tested) is loaded onto the sequencing chip through the Salus Pro system microfluidic module, it is first hybridized with the primers fixed on the surface of the sequencing chip. After the hybridization is completed, a cleaning reagent is introduced to wash away the unhybridized template.

[0078] 2) Extend the template complementary chain

[0079] First, the amplification cleaning solution is pumped into the sequencing chip, and then the amplification extension solution (a mixture of BST polymerase and dNTP) is pumped into the sequencing chip to extend the hybridized library template chain with the surface primer to fix the library information to the surface of the solid phase sequencing chip.

[0080] 3) Bridge Amplification

[0081] Isothermal bridge amplification was performed on the library template chain of the sequencing chip: the amplification parameters were set on the system, the chip surface temperature was kept constant at 55°C, and the system executed the process of "pumping formamide (denaturation) - pumping amplification cleaning solution (annealing) - pumping amplification extension solution (extension)", and repeated 30 times to amplify the template cluster to be tested (bridge complementary chain).

[0082] 4) Linearization and blocking of the first template strand to be tested

[0083] Linearization: Pump in the cleaning solution to clean the sequencing chip, then pump in the first linearization reagent prepared with USER enzyme, react at 37°C for 30 minutes, during which USER enzyme recognizes and removes the modified restriction site ideoxyU in the surface P5 primer. At this time, the P5 extended chain (the second template chain to be tested) in the double-stranded amplification cluster is cut off, and after washing with formamide, the remaining P7 extended chain (the first template chain to be tested) is connected to the surface of the sequencing chip;

[0084] Blocking: Pump in terminal transferase (TDT) and ddNTP mixture at the same time and react at 37°C for 30 minutes to block the unused primers on the surface and the 3' end of the first template chain to be tested to prevent the introduction of erroneous signals during sequencing.

[0085] 5) SBS sequencing of the first template strand to be tested

[0086] Sequencing primer SP1 hybridization: After formamide is pumped into the sequencing chip for elution, a cleaning solution is pumped in for cleaning, and then sequencing primer SP1 is pumped in for reaction at 55°C for 15 minutes. After hybridization is completed, a cleaning solution is pumped in for cleaning.

[0087] The base sequence of SP1 primer sequence (SEQ ID NO: 3):

[0088] 5'-ACACTCTTTCCCTACACGACCGCTCTTCCGATCT-3'.

[0089] SBS sequencing: The sequencing read length parameters were set on the system. The system executed the process of “pumping in a mixture of 9N mutant enzyme, fluorescent marker and 3' blocked dNTP (synthesis reaction) – the system optical module collected the fluorescence signal (signal collection) – pumping in the excision reagent to excise the blocking group and the fluorescent group (excision reaction)”, which was repeated 75 times for first-strand sequencing. The sequencing read length was 75 bp.

[0090] 3. Generation and sequencing of the second test template strand

[0091] like Figure 1 and Figure 2 As shown, the steps include:

[0092] 1) Second template strand to be tested is extended and generated

[0093] After the last round of SBS sequencing of the first template chain to be tested is completed, the amplification cleaning solution is pumped in, and then the amplification extension solution (a mixture of BST polymerase and dNTP) is pumped in. The surface temperature is set to 55°C and the reaction is allowed to stand for 8 minutes. At this time, the sequencing chain grown by the original SP1 sequencing primer can continue to extend to the last base, that is, to generate the second template chain to be tested.

[0094] 2) The second template strand to be tested is fixed on the surface of the sequencing chip

[0095] Introduction of butyne-modified dNTP at the end: After the extension of the second template chain to be tested is completed, a mixture of terminal transferase (TDT) and a 3'-end butyne-modified dNTP is pumped in, and the reaction is carried out at 37°C for 30 minutes, and then a cleaning solution is pumped in. At this time, the 3' end of the second template chain to be tested is a butyne-modified dNTP, and its structural formula is shown in Formula III:

[0096]

[0097] Fixation of the second template chain to be tested on the surface: Pump in Cu(I) catalyst and react at 60°C for 60 minutes. At this time, under the catalysis of Cu(I), the alkyne group in the dNTP introduced in step 2) above undergoes cycloaddition with the azide group on the surface (azide-alkyne cycloaddition reaction), thereby fixing the 3' end of the generated second chain on the surface of the sequencing chip. Among them, the structure of the dNTP modified with butyne and the reaction formula with the surface are:

[0098]

[0099] 4) Chaining of the second test template

[0100] After pumping in the cleaning solution to clean the chip, pump in the second linearization reagent prepared with FPG enzyme, and react at 37°C for 30 minutes. During the reaction, the FPG enzyme recognizes and removes the modified restriction site i8oxodG in the surface P7 primer. At this time, the P7 extension chain (the first template chain to be tested) in the double-stranded amplification cluster is cut off. After pumping in formamide for washing, the newly generated second template chain to be tested is connected to the chip surface.

[0101] 5) SBS sequencing of the second template strand to be tested

[0102] Sequencing primer SP2 hybridization: pump in formamide for elution, then pump in cleaning solution for cleaning, then pump in sequencing primer SP2, react at 55°C for 15 minutes, and after hybridization is completed, pump in cleaning solution for cleaning;

[0103] The base sequence of SP2 primer sequence (SEQ ID NO: 4):

[0104] 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'.

[0105] SBS sequencing: The sequencing read length parameters were set on the system. The system executed the process of “pumping in 9N mutant enzyme, fluorescent marker and 3' blocked dNTP mixture (synthesis reaction) – the system optical module collected the fluorescence signal (signal collection) – pumping in the excision reagent to excise the blocking group and the fluorescent group (excision reaction)”, and repeated 75 times for second-chain sequencing. The sequencing read length was 75 bp.

[0106] Example 2

[0107] 1. Preparation of sequencing chip with modified P5 and P7 primers

[0108] 1) obtaining a sequencing chip chemically modified with trans-cyclooctene (TCO);

[0109] 2) The primers P5 and P7 were prepared into primer solutions with a concentration of 5uM each using 3×SSC solution and introduced into the chip flow channel. The primers were heated in a 60°C oven for 1 hour. The 5' ends of the two primers were modified with 1,2,4-triazine. The 1,2,4-triazine modification at the 5' end reacted with trans-cyclooctene (TCO) on the chip surface to fix the primers on the chip surface.

[0110] 3) After the reaction is completed, each flow channel is cleaned with 3×SSC solution and set aside; the obtained sequencing chip can be used for subsequent amplification sequencing.

[0111] Wherein the P5 primer and the P7 primer are the same as those in Example 1.

[0112] 2. Amplification of the library to be tested on the sequencing chip and first-strand sequencing

[0113] The specific steps are the same as the corresponding steps in Example 1.

[0114] 3. Generation and sequencing of the second test template strand

[0115] like Figure 1 and Figure 2 As shown, the steps include:

[0116] 1) Second template strand to be tested is extended and generated

[0117] After the last round of SBS sequencing of the first template chain to be tested is completed, the amplification cleaning solution is pumped in, and then the amplification extension solution (a mixture of BST polymerase and dNTP) is pumped in. The surface temperature is set to 55°C and the reaction is allowed to stand for 8 minutes. At this time, the sequencing chain grown by the original SP1 sequencing primer can continue to extend to the last base, that is, to generate the second template chain to be tested.

[0118] 2) The second template strand to be tested is fixed on the surface of the sequencing chip

[0119] Introduction of 1,2,4-triazine-modified dNTP at the end: After the extension of the second template chain to be tested is completed, terminal transferase (TDT) and a mixture of dNTPs with 1,2,4-triazine modified at the 3' end are pumped in. After reacting at 37°C for 30 minutes, a cleaning solution is pumped in. At this time, the 3' end of the second template chain to be tested is a dNTP with 1,2,4-triazine modified, and its structural formula is shown in Formula IV:

[0120]

[0121] Fixation of the second template chain to be tested on the surface: The reaction conditions are set at 60°C for 60 minutes. The 1,2,4-triazine in the dNTP introduced in step 2) above undergoes a reverse electron demand Diels-Alder coupling reaction with the trans-cyclooctene (TCO) on the surface, thereby fixing the 3' end of the generated second template chain to be tested on the surface of the sequencing chip. The structure of the dNTP modified with 1,2,4-triazine and the reaction formula with the surface are:

[0122]

[0123] 4) Linearization of the second template strand to be tested

[0124] After pumping in the cleaning solution to clean the chip, pump in the second linearization reagent prepared with FPG enzyme, and react at 37°C for 30 minutes. During the reaction, the FPG enzyme recognizes and removes the modified restriction site i8oxodG in the surface P7 primer. At this time, the P7 extension chain (the first template chain to be tested) in the double-stranded amplification cluster is cut off. After pumping in formamide for washing, the newly generated second template chain to be tested is connected to the chip surface.

[0125] 5) SBS sequencing of the second template strand to be tested

[0126] Sequencing primer SP2 hybridization: pump in formamide for elution, then pump in cleaning solution for cleaning, then pump in sequencing primer SP2, react at 55°C for 15 minutes, and after hybridization is completed, pump in cleaning solution for cleaning;

[0127] The base sequence of SP2 primer sequence (SEQ ID NO: 4):

[0128] 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'.

[0129] SBS sequencing: The sequencing read length parameters were set on the system. The system executed the process of “pumping in 9N mutant enzyme, fluorescent marker and 3' blocked dNTP mixture (synthesis reaction) – the system optical module collected the fluorescence signal (signal collection) – pumping in the excision reagent to excise the blocking group and the fluorescent group (excision reaction)”, and repeated 75 times for second-chain sequencing. The sequencing read length was 75 bp.

[0130] Example 3

[0131] The difference between this embodiment and embodiment 1 is that:

[0132] The sequencing chip adopts an acetylene-modified sequencing chip;

[0133] The second test template chain end is introduced with an azide-modified dNTP, and the steps include: after the second test template chain is extended, pumping in a terminal transferase (TDT) and a dNTP mixture with an azide-modified 3' end, reacting at 37° C. for 30 minutes, and then pumping in a cleaning solution. At this time, the 3' end of the second test template chain is an azide-modified dNTP, and its structural formula is shown in Formula V:

[0134]

[0135] Fixation of the second template chain to be tested on the surface: Pump in Cu(I) catalyst and react at 60°C for 60 minutes. At this time, under the catalysis of Cu(I), the alkyne group in the dNTP introduced in step 2) above undergoes cycloaddition with the azide group on the surface (azide-alkyne cycloaddition reaction), thereby fixing the 3' end of the generated second chain on the surface of the sequencing chip. Among them, the structure of the dNTP modified with butyne and the reaction formula with the surface are:

[0136]

[0137] Example 4

[0138] The difference between this embodiment and embodiment 2 is that:

[0139] The sequencing chip is a sequencing chip modified with 1,2,4,5-tetrazine derivatives;

[0140] The second test template chain end is introduced with 1-methylcyclopropene modified dNTP, and the steps include: after the second test template chain is extended, pumping in terminal transferase (TDT) and a dNTP mixture with 1-methylcyclopropene modified at the 3' end, reacting at 37° C. for 30 minutes, and then pumping in a cleaning solution, at which time the 3' end of the second test template chain is a 1-methylcyclopropene modified dNTP, and its structural formula is shown in Formula VI:

[0141]

[0142] Fixation of the second template chain to be tested on the surface: The reaction conditions are set at 60°C for 60 minutes. The 1-methylcyclopropene in the dNTP introduced in step 2) above undergoes a reverse electron demand Diels-Alder coupling reaction with the 1,2,4,5-tetrazine derivative on the surface, thereby fixing the 3' end of the generated second template chain to be tested on the surface of the sequencing chip. The structure of the dNTP modified with 1-methylcyclopropene and the reaction formula with the surface are:

[0143]

[0144] Performance Testing

[0145] 1. Sequencing accuracy experiment

[0146] The original images collected by the SBS sequencing of the first template strand to be tested and the SBS sequencing of the second template strand to be tested in Example 1 and Example 2 were subjected to base calling using the NGS software provided by the salus Pro system, and finally the number of clusters (TotalReads) and base quality scores of the sequencing of the first template strand to be tested (Read1) and the sequencing of the second template strand to be tested (Read2) were obtained. The results are shown in Tables 1 and Figures 3 to 8 shown.

[0147] Table 1 Statistics of first and second strand sequencing results

[0148]

[0149] from Figures 3 to 5 As can be seen from Table 1, in the double-end sequencing method of Example 1 of the present invention, the cluster morphology of the original signal of the first template strand to be tested and the second template strand to be tested in SBS sequencing is consistent and highly uniform ( Figure 3 ). At the same time, the sequencing signals of the first test template chain and the second test template chain in each round are similar, and their signal values ​​meet the requirements of base recognition ( Figure 4); In addition, the base quality scores of the first template strand to be tested and the second template strand to be tested have the same changing trend, and the base quality scores are also similar ( Figure 5 ); The base contents of the two chains are close, with Q30 percentages of approximately 86.8% and 86.4% respectively, with high sequencing quality and high sequencing accuracy

[0150] from Figures 6 to 8 As can be seen from Table 1, in the double-end sequencing method of Example 2 of the present invention, the cluster morphology of the original signal of the first template strand to be tested and the second template strand to be tested in SBS sequencing is consistent and highly uniform ( Figure 6 ). At the same time, the sequencing signals of the first test template chain and the second test template chain in each round are similar, and their signal values ​​meet the requirements of base recognition ( Figure 7 ); In addition, the base quality scores of the first template strand to be tested and the second template strand to be tested have the same changing trend, and the base quality scores are also similar ( Figure 8 ); the base contents of the two chains are close, with Q30 percentages of approximately 86.2% and 85.7% respectively, with high sequencing quality and high sequencing accuracy.

[0151] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A double-end sequencing method, characterized in that: The following steps are involved: S1, loading a double-stranded template polynucleotide to be tested on a sequencing chip with fixed primers fixed on the surface for amplification, to obtain a double-stranded polynucleotide to be tested fixed on the surface of the sequencing chip; the surface of the sequencing chip is modified with a first active group, and the fixed primers include a P5 primer and a P7 primer having different restriction sites; S2, selectively removing the second test template strand extended by the P5 primer in the test polynucleotide double strand, leaving the first test template strand extended by the P7 primer, and then performing a first sequencing on the first test template strand; The first sequencing comprises: performing a first SBS sequencing on the first template strand to be tested using the SP1 primer, and obtaining a sequencing strand; S3, using the first test template strand as a template, extending and synthesizing an extended strand on the sequencing strand to obtain a second test template strand, wherein the second test template strand is sequentially composed of an SP1 primer, a sequencing strand, and an extended strand; S4, connecting the end of the second test template chain synthesized in step S3 with a dNTP modified with a second active group; performing a click reaction between the second active group and the first active group to fix the second test template chain on the surface of the sequencing chip; selectively removing the first test template chain, and then performing a second sequencing on the second test template chain; The fixed primer is covalently fixed to the surface of the sequencing chip via the 5' end; and, The second template strand to be tested is fixed to the surface of the sequencing chip via the 3' end; The click reaction includes an azide-alkyne cycloaddition reaction or a reverse electron demand Diels-Alder coupling reaction.

2. The double-end sequencing method according to claim 1, characterized in that: The selective excision of the second test template chain comprises: performing enzyme cleavage on the enzyme cleavage site of the P5 primer, and then denaturing and eluting to remove the second test template chain complementary to the first test template chain; and / or, The selective excision of the first test template chain includes: performing enzyme cleavage on the enzyme cleavage site of the P7 primer, and then denaturing and eluting to remove the first test template chain that is complementary hybridized with the second test template chain.

3. The double-end sequencing method according to claim 1, characterized in that: In step S4, connecting the dNTP modified with the second active group to the end of the second template chain to be tested includes: adding terminal transferase and the dNTP modified with the second active group to the second template chain to be tested synthesized in step S3, so that the end of the second template chain to be tested is connected to the dNTP modified with the second active group.

4. The double-end sequencing method according to claim 1, characterized in that: The structural formula of the dNTP modified with the second active group is shown in Formula I; Formula I Wherein, the R1 is selected from azido, 1-methylcyclopropenyl, C2-C4 alkynyl or C2-C4 alkenyl, and Base is a base.

5. The double-end sequencing method according to claim 1, characterized in that: The structural formula of the dNTP modified with the second active group is shown in Formula II; Formula II Wherein, R2 is selected from 1,2,4-triazine, 1-methylcyclopropene, trans-cyclooctenyl, azido, C2-C4 alkynyl or C2-C4 alkenyl; R3 is selected from H, C1-C3 alkyl, and Base is a base.

6. The double-end sequencing method according to claim 1, characterized in that: The first reactive group is selected from an azido group, a C2-C4 alkynyl group, a trans-cyclooctenyl group, a 1,2,4,5-tetrazine group or a 1-methylcyclopropene group.

7. The double-end sequencing method according to claim 1, characterized in that: The P5 includes an enzyme cleavage site ideoxyU.

8. The double-end sequencing method according to claim 1 or 7, characterized in that: The P7 includes an enzyme cleavage site i8oxodG.

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