Library linker design to enhance sequencing throughput

Abstract

The invention provides a method for establishing a sequencing library, the sequencing library, a sequencing method and a kit for establishing the sequencing library or sequencing. The method for establishing the sequencing library comprises the following steps: performing digestion treatment on a to-be-detected sample connected with a joint under the action of exonuclease, so as to obtain the sequencing library. Wherein the 5'end of the joint is not subjected to phosphorylation modification, and the 3 'end of the joint is connected with the 5' end of the sample to be detected.

Description

Library adapter design to improve sequencing throughput Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for establishing a sequencing library, and more specifically, relates to a sequencing library, a sequencing method, and a kit for constructing a sequencing library or sequencing. Background Art

[0002] Nucleic acid sequencing has become an indispensable and important research tool in the field of life sciences. The accompanying genomics technology, based on large-scale sequencing data, has also played a vital role in various research and application areas, such as tracing the causes of complex diseases and dynamically monitoring their development, targeted breeding of economically viable crops and animals, and the research and protection of diverse biological genetic resources.

[0003] However, a prerequisite for such research is the ability to utilize high-throughput parallel sequencing to obtain highly accurate, complete nucleic acid sequences. Current mainstream sequencing-by-synthesis technologies offer high accuracy, with most bases correctly identified reaching over 99.9% accuracy. However, these technologies suffer from short read lengths, making it difficult to recover the complete target nucleic acid sequence using either resequencing or de novo assembly.

[0004] Therefore, how to obtain long-range information from nucleic acid sequences has become a hot topic in sequencing technology research. Currently, there are two major companies offering commercial long-read sequencing solutions: Pacific Bioscience (Pacbio) in the United States, which offers single-molecule real-time sequencing, and Oxford Nanopore Technologies (ONT) in the United Kingdom, which offers nanopore sequencing. Furthermore, international companies such as Quantum SI and Genia, as well as domestic companies such as Qitan, Jinshi, and Anxuyuan, have entered this technology field and released prototypes.

[0005] With the advancement of science and technology, current clinical sample variation detection is no longer satisfied with detecting only small-range variations (single nucleotide mutations and small deletion / insertion mutations). Some genetic abnormalities caused by large-scale structural variations are gradually being analyzed. Since structural variation detection is more sensitive to sequencing read length, long-read sequencing technology will gradually shift from the field of scientific and technological services to the field of clinical testing, and the requirements for the accuracy and cost control of sequencing technology will also increase accordingly.

[0006] In terms of accuracy, Pacbio launched the Hi-Fi sequencing method based on circular consensus sequencing in 2019 (Figure 1). Repeating the reading five times can achieve an average sequencing accuracy of more than 99%, but because it requires multiple repeated sequencing of the same molecule, the cost is relatively high.

[0007] To reduce costs, Oxford Nanopore's newly released PromethION 48 system offers a cost per Gb of $2-16, approaching the cost of sequencing by synthesis, a widely used method in the market. While the accuracy of this system still lags behind Pacbio's circularization consensus sequencing method, according to recently released data from Oxford Nanopore, it has reached 98.4%, a significant improvement over earlier Nanopore data.

[0008] The current advantage of nanopore sequencing solutions is its low cost, but the sequencing accuracy still has room for improvement. Unlike conventional synthesis sequencing, nanopore sequencing libraries generally have longer inserts, ranging from a few thousand bases to a million bases. Therefore, they have fewer double-stranded ends than short insert libraries, and the efficiency of adapter connection is relatively low. Because nanopore sequencing adapters are generally coupled with the rate-control protein required for sequencing, alcohol-containing cleaning reagents cannot be used during magnetic bead or column purification after connection, making it difficult to remove the adapters during the purification step, resulting in the detection of non-target fragments during the sequencing process and a decrease in sequencing accuracy.

[0009] Therefore, there is a need in the art to develop a method for optimizing the library construction process to reduce the amount of residual adapters in the library, thereby reducing the detection of invalid sequencing adapters by sequencing channels during the sequencing process.

[0010] Summary of the Invention

[0011] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0012] In order to solve the problem of rate-controlling protein denaturation caused by the use of alcohol detergents in the column purification process and reduce the detection of invalid sequencing adapters in the sequencing channel, the inventors found that, on the one hand, a nuclease can be added during the library construction process to remove residual adapters in the library. During the purification process, no alcohol detergent is used, and only magnetic beads or columns are used for purification. On the other hand, by not performing phosphorylation modification on the 5' end of the adapter, this not only avoids the detection of non-target fragments during the sequencing process and improves the sequencing accuracy, but also optimizes the use efficiency of anchor sequences during the sequencing process, increases sequencing throughput, and obtains high-quality sequencing reads in a short time.

[0013] Based on the above findings, in a first aspect of the present invention, the present invention proposes a method for establishing a sequencing library. According to an embodiment of the present invention, the method comprises: digesting a sample to be tested connected to a connector under the action of a nuclease to obtain the sequencing library. Wherein, the 5' end of the connector does not have a phosphorylation modification, and the 3' end of the connector is connected to the 5' end of the sample to be tested. According to the method of an embodiment of the present invention, on the one hand, by introducing a nuclease to degrade the fragment to be tested that has not been completely connected, the product of incomplete connector connection, and the unconnected connector, the number of abnormal sequencing is reduced, and the sequencing accuracy and sequencing throughput are improved. On the other hand, by using a connector with no phosphorylation modification at the 5' end and treating the connected product with a nuclease, the connector itself is avoided from connecting to form a dimer, and the free connector that is not connected to the two ends of the inserted fragment to be tested is digested into a single chain, thereby reducing the residual connector sequence that may be sequenced or reducing the time it occupies the sequencing channel, thereby improving the sequencing throughput. At the same time, since the chain without phosphorylation modification at the 5' end will be further degraded by nuclease, exposing the anchor sequence binding site (because the 5' end cannot be connected to the 3' end of another single chain when it is not phosphorylated, it will be recognized and digested by nuclease), and the anchor sequence itself will not be covalently bound to the library to be tested, after the motor protein passes through the anchor sequence binding site, the anchor sequence will be stripped from the sequencing library itself, thereby realizing the reuse of the anchor sequence and improving the library capture efficiency.

[0014] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0015] According to an embodiment of the present invention, the sample to be tested connected to the adapter has a gap, and the gap is located between the 5' end of the adapter and the 3' end of the sample to be tested.

[0016] According to an embodiment of the present invention, the method further comprises subjecting the obtained digestion product to a detwisting treatment.

[0017] According to an embodiment of the present invention, the unwinding process is performed under the action of a helicase.

[0018] According to an embodiment of the present invention, the helicase moves along the DNA from the 5' end to the 3' end, and the exonuclease is a 5'->3' exonuclease.

[0019] According to an embodiment of the present invention, the 5'->3' exonuclease recognizes double-stranded DNA, and the exonuclease includes at least one selected from T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, and Exonuclease VIII (truncated).

[0020] According to an embodiment of the present invention, the exonuclease is preferably T7 exonuclease.

[0021] According to an embodiment of the present invention, the helicase moves along the DNA from the 3' end to the 5' end, and the exonuclease is a 3'->5' exonuclease.

[0022] According to an embodiment of the present invention, the 3'->5' exonuclease recognizes double-stranded DNA, and the exonuclease includes at least one selected from Exonuclease III, Exonuclease IX, and Exonuclease X.

[0023] According to an embodiment of the present invention, the exonuclease recognizes single-stranded DNA.

[0024] According to an embodiment of the present invention, the method for establishing a sequencing library further includes performing a degradation-resistant modification on the 5' end and / or 3' end of the sample to be tested to which the adapter is connected. For digestion of the ligation product using exonucleases that can recognize and digest the 5' end of single-stranded DNA (e.g., T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, Exonuclease VIII (truncated)), the 5' end of the single-stranded DNA, for example, the 5' end of the sequence to be tested to which the Y-type adapter is connected, can be chemically modified in advance to resist degradation by nucleases.

[0025] According to an embodiment of the present invention, the anti-degradation modification includes at least one selected from phosphate modification, 2'-OH modification (RNA base), 2'-F modification, LNA locked nucleotide modification and PNA peptide nucleic acid modification.

[0026] According to an embodiment of the present invention, the digestion treatment is performed at 37° C. with a ratio of ligation product to T7 exonuclease of 44:1 for 4 to 6 minutes.

[0027] According to an embodiment of the present invention, the connector is a Y-shaped connector or a non-Y-shaped connector.

[0028] According to an embodiment of the present invention, the non-Y-shaped linker has at least one of the following structures: complete complementary double strand, complementary double strand-non-complementary single strand-complementary double strand, 5' protruding single strand-complementary double strand, 3' protruding single strand-complementary double strand.

[0029] According to an embodiment of the present invention, the method for establishing a sequencing library further includes purifying the digestion product.

[0030] According to an embodiment of the present invention, the purification process uses Ampure XP magnetic beads for purification.

[0031] According to an embodiment of the present invention, the sample to be tested connected with the connector is obtained by:

[0032] (1) Perform end repair and A-addition treatment (adding poly-A tail) on the sample to be tested;

[0033] (2) The product treated with A is subjected to a linker connection treatment to obtain the sample to be tested connected with a linker.

[0034] According to an embodiment of the present invention, after the end repair and A addition treatment and before the ligation treatment, the process further includes performing a first purification treatment on the A addition treatment product.

[0035] According to an embodiment of the present invention, after the ligation process, the method further includes performing a second purification process on the ligation product.

[0036] According to an embodiment of the present invention, the sample to be tested is a DNA sample.

[0037] According to an embodiment of the present invention, before performing end-repair and A-addition treatment on the sample to be tested, the method further includes performing fragmentation treatment on the sample to be tested.

[0038] In a second aspect, the present invention further provides a sequencing library. According to an embodiment of the present invention, the sequencing library is a sequencing library of a nucleic acid sample obtained according to a method for establishing a sequencing library according to an embodiment of the present invention. The inventors have found that the sequencing library obtained using this method significantly reduces the number of unligated test fragments, unligated free adapters, and incomplete adapter ligation products, thereby increasing the utilization rate of anchor sequences. Furthermore, the above steps are simple to perform, significantly improve sequencing accuracy, have good reproducibility, are low-cost, and have a higher sequencing throughput.

[0039] In a third aspect, the present invention further provides a sequencing method. According to an embodiment of the present invention, the method comprises mixing the aforementioned sequencing library with an anchor sequence, wherein at least a portion of the anchor sequence is complementary to at least a portion of one strand of the adapter. The mixed product is sequenced to obtain the sequence of the sample to be tested.

[0040] According to an embodiment of the present invention, the sequencing is performed on a nanopore sequencing platform.

[0041] The inventors found that this method can efficiently determine the sequence information of nucleic acid samples, and has high sensitivity, high accuracy, good repeatability and high sequencing throughput.

[0042] In a fourth aspect of the present invention, the present invention further provides a kit for constructing a sequencing library or sequencing. According to an embodiment of the present invention, the kit includes reagents, including a nuclease exonuclease, for digesting a sample to be tested to which a linker is connected. The inventors have discovered that by utilizing the kit of the present invention, in combination with the above-mentioned method for establishing a sequencing library, sequencing library, and sequencing method, a high-quality sequencing library can be obtained, and can be effectively applied to a high-throughput sequencing platform to thereby determine the nucleic acid sequence information of a nucleic acid sample, and the obtained information has high accuracy and high sequencing throughput.

[0043] According to an embodiment of the present invention, the kit further includes at least one of the following additional technical features:

[0044] According to an embodiment of the present invention, the reagent further includes a first reagent, and the first reagent is suitable for fragmenting the sample to be tested.

[0045] According to an embodiment of the present invention, the reagent further includes a second reagent, and the second reagent is suitable for performing end-repair and A addition treatment on the sample to be tested.

[0046] According to an embodiment of the present invention, the reagent further includes a third reagent, and the third reagent is suitable for performing a connector connection process on the sample to be tested.

[0047] According to an embodiment of the present invention, the kit further comprises a linker, and the 5' end of the linker is not phosphorylated.

[0048] According to an embodiment of the present invention, the exonuclease includes at least one of T7 exonuclease, Lambda exonuclease, T5 exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X.

[0049] According to an embodiment of the present invention, the kit further includes an anchor sequence, at least a portion of which is complementary to at least a portion of one strand of the adapter. The anchor sequence can then be attached to one strand of the adapter through complementary base pairing, thereby bringing the adapter-attached sequence to the vicinity of the nanopore sequencing well for sequencing.

[0050] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0052] FIG1 is a diagram illustrating the Pacbio circularization consensus sequencing principle according to an embodiment of the present invention;

[0053] FIG2 is a flow chart of library construction including an exonuclease step according to an embodiment of the present invention;

[0054] FIG3 is a diagram of a sequencing process using a 5' unphosphorylated adapter and a 5'->3' exonuclease according to an embodiment of the present invention;

[0055] FIG4 is a graph showing the purity of the purified protein according to an embodiment of the present invention; FIG4 is a graph showing the purity of the purified product using HPLC, and FIG4 is a graph showing the purity of the purified product using SDS-PAGE;

[0056] FIG5 is a comparison diagram of library bands before and after enzyme digestion according to an embodiment of the present invention;

[0057] FIG6 is a comparison result of the number of single-channel sequencing signals after library construction and sequencing with and without phosphorylation of the adapter 5′ according to an embodiment of the present invention.

[0058] Detailed Description of the Invention

[0059] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0060] It should be noted that the terms "first," "second," and "third" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first," "second," or "third" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0061] Methods for establishing sequencing libraries

[0062] According to one aspect of the present invention, a method for establishing a sequencing library is provided. According to an embodiment of the present invention, with reference to FIG2 , the method comprises the following steps: initial nucleic acid molecule fragmentation (optional), end repair and A addition, adapter ligation, nuclease treatment, and anchor sequence annealing. The specific implementation is as follows:

[0063] According to an embodiment of the present invention, the first step is to selectively fragment the genomic DNA sequence to obtain fragmented genomic DNA molecules, which can improve the library capture rate during sequencing. The second step is to end-repair the fragmented genomic DNA molecules and add "A". The third step is to purify the genomic DNA after "A" addition to obtain a relatively pure sample, so as to increase the accuracy and sequencing throughput during sequencing. The fourth step is to perform adapter ligation on the purified sample. According to an embodiment of the present invention, adapter ligation refers to mixing adapters with T4 DNA ligase (NEB, E6057AVIAL) to obtain a sample after adapter ligation. The fifth step is to purify the adapter ligation sample. A higher purity sample is extremely important for the accuracy and data volume of sequencing. The sixth step is to digest the purified ligation product with the action of an exonuclease to degrade the test fragment that has not been completely ligated, the free adapter strand that is not connected to the two ends of the test insert, and the product of incomplete adapter ligation. This reduces the time that the sequencing channel is occupied by the undesired library and thus improves sequencing throughput. According to an embodiment of the present invention, exonuclease can be selected from at least one of T7exonuclease, T5exonuclease, Lambda exonuclease, Exonuclease VI, and Exonuclease VIII (truncated). In the seventh step, the sample treated with the exonuclease is subjected to product purification to obtain a preliminary library sample. In the eighth step, the preliminary library sample obtained is incubated with the helicase Dda protein to finally obtain a library that can be used for sequencing. According to an embodiment of the present invention, the helicase moves from the 5' end to the 3' end along the direction of movement of the DNA.

[0064] According to embodiments of the present invention, this method can efficiently prepare sequencing samples, and the resulting sequencing library can be effectively applied to a high-throughput sequencing platform, thereby effectively determining the nucleic acid sequence information of the library samples. Furthermore, the inventors surprisingly discovered that the method for preparing a sequencing library of the present invention is simple and easy to operate, with an easily standardized and scalable operational process, low cost, high sensitivity, high accuracy, and good reproducibility.

[0065] Acquisition of sequencing libraries

[0066] The library of the sample to be tested is obtained by the above-mentioned method for establishing a sequencing library.

[0067] Sequencing methods

[0068] According to a specific embodiment of the present invention, a sequencing library is constructed using a 5' unphosphorylated linker and a 5'->3' exonuclease, and the library sample to be tested is placed on a nanopore sequencing platform for on-machine sequencing. Among them, the anchor sequence captures the sequencing library near the nanopore for sequencing. By adopting electrophoresis technology, sequencing is achieved by driving single bases through the nanopore one by one with the help of electrophoresis. After sequencing begins, the anchor sequence is released and the next library can be captured (Figure 3). Since the charged properties of individual ATCG bases are different, the type of base passed can be detected by the difference in electrical signals, thereby achieving sequencing.

[0069] Motor proteins

[0070] Motor protein (helicase in Example 2 of the present invention) refers to a type of protein distributed inside or on the surface of cells, which is responsible for the macroscopic movement of a part of the substance in the cell or the entire cell.

[0071] Primers

[0072] The term "primer" as used herein refers to an oligonucleotide that can complementarily pair with a template and, under the action of DNA polymerase, synthesize a DNA strand complementary to the template. Primers can be natural RNA, DNA, any form of natural nucleotides, or even non-natural nucleotides such as LNA or ZNA.

[0073] iSpC3

[0074] iSpC3 is a three-hydrocarbon carbon chain that is commonly used as a spacer in oligonucleotide chains.

[0075] iSp18

[0076] iSp18 is an 18-atom-long hexaethylene glycol chain that is commonly used as a spacer in oligonucleotide chains.

[0077] Exonucleases

[0078] Exonucleases are enzymes that degrade nucleotides one by one, starting from the ends of polynucleotide chains. Based on their specificity for substrate secondary structures, they are divided into three categories: ① Exonucleases that act on single strands, such as E. coli exonuclease I and E. coli exonuclease VII. ② Exonucleases that act on double strands, such as E. coli exonuclease III, bacteriophage exonucleases, and T7 phage gene VI exonuclease.

[0079] The present invention will be further explained below with reference to specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0080] Example 1: Preparation of linker sequences

[0081] In this example, the linker sequence was prepared by annealing chemically synthesized SEQ ID NO. 1 and SEQ ID NO. 2. The specific implementation method is as follows:

[0082] 1. Order the primer sequences for SEQ ID NO. 1 and SEQ ID NO. 2 (Sangon Biotech). Dissolve the primers for SEQ ID NO. 1 and SEQ ID NO. 2 in TE buffer (pH 8) according to the manufacturer's instructions to a stock solution with a final concentration of 100 μM. Then, take 10 μL of each stock solution and add 40 μL of TE buffer (pH 8) to dilute the solution to a final concentration of 20 μM.

[0083] 2. Take 30 μL of the working solution of the primer SEQ ID NO. 1 and 30 μL of the working solution of the primer SEQ ID NO. 2 obtained by dilution in the previous step, mix them together, and thoroughly vortex them. Heat them to 70°C in a thermal cycler and incubate them for 10 minutes. Then, cool them to 25°C at a rate of 0.1°C / s and continue incubating for half an hour to obtain a 10 μM annealed linker solution. This produces a 5'-terminal phosphorylated linker, named Ad3, and store it in a -20°C refrigerator.

[0084] 3. Take 30 μL of the working solution of SEQ ID NO. 1 primer and 30 μL of the working solution of SEQ ID NO. 3 primer obtained by dilution in the previous step, mix them together, and use a vortex to shake and mix thoroughly. Heat to 70°C in a thermal cycler and incubate for 10 minutes. Then, cool to 25°C at a rate of 0.1°C / s and continue incubation for half an hour to obtain a 10 μM annealed linker solution. This prepares a linker with no phosphorylation modification at the 5' end (SEQ ID NOs: 1 and 3). The linker product is named Ad3-no pho and stored in a refrigerator at -20°C.

[0085] 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3' (X=iSpC3; Y=iSp18; SEQ ID NO. 1).

[0086] 5'pho-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3' (SEQ ID NO. 2).

[0087] 5'-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3' (SEQ ID NO. 3).

[0088] Example 2. Cloning, expression and purification of helicase Dda

[0089] In this example, helicase Dda (amino acid sequence shown in SEQ ID NO. 5) was prepared by recombinant expression in Escherichia coli, and the helicase was used as a motor protein.

[0090] 1. The full-length Dda cDNA sequence (nucleotide sequence shown in SEQ ID NO. 4) was ordered from Sangon Biotechnology and ligated into the pET.28a(+) plasmid. The vector was digested with double enzymes at the Nde1 and Xho1 sites to express the Dda protein with a 6×His tag and a thrombin cleavage site at the N-terminus.

[0091] 2. Transform the cloned PET.28a(+)-Dda plasmid into ArcticExpress (DE3) competent bacteria (Tolo Biotech, 96183-02) or its derivatives. Pick a single colony and transfer it to 5 mL of LB medium containing kanamycin for propagation. Incubate at 37°C with shaking overnight. Then transfer it to 1 L of LB medium (containing kanamycin) and incubate at 37°C with shaking until the OD 600 The pH value was 0.6-0.8, the temperature was lowered to 16°C, and IPTG was added to a final concentration of 500 μM to induce Dda expression overnight.

[0092] 3. Prepare five buffer solutions according to the following formula:

[0093] Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

[0094] Buffer B: 20 ​​mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0095] Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

[0096] Buffer D: 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;

[0097] Buffer E: 20 mM Tris-HCl pH 7.5, 100 mM NaCl.

[0098] 4. Collect the Dda cells expressed in step 2. Resuspend the cells in buffer A from step 3, disrupt the cells using a cell disruptor, and centrifuge to collect the supernatant. Mix the supernatant with Ni-NTA filler previously equilibrated with buffer A and allow to bind for 1 hour. Collect the filler and wash it extensively with buffer A until all impurities are washed out. Then, add buffer B to the filler to elute the Dda. Pass the eluted Dda through a desalting column equilibrated with buffer C to perform a buffer exchange. Then, add an appropriate amount of thrombin (Yishen Bio, 20402ES05), and add the mixed sample to a ssDNA cellulose filler equilibrated with buffer C. Digest and bind overnight at 4°C. Collect the ssDNA cellulose filler, wash it 3-4 times with buffer C, and then elute it with buffer D. Pass the protein concentrate purified from the ssDNA cellulose through a Superdex 200 column (Sigma, GE28-9909-44) using buffer E. The target protein was collected, concentrated, and frozen. The purified protein concentration was quantified using Nanodrop. Protein purity was also tested using HPLC and SDS-PAGE electrophoresis. The results are shown in Figure 4.

[0099]

[0100]

[0101] MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGGTGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKCRVL ICDEVSMYDRKLFKILLSTIPPWCTIIGIGDNKQIRPVEPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWNYDKVVDGHGVRGFTGDTALRDFMVNYFSI VKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYR EKIKIISSDEELYKFNLFLAKTAETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV*(* indicates termination; SEQ ID NO.5)

[0102] Example 3 Construction and purification of the helicase (Dda) and linker (Ad3, Ad3-no pho) complex

[0103] This example takes the construction and purification of the Dda and Ad3 complex as an example, and the specific steps are as follows:

[0104] 1. Construction of the Dda-Ad3 Complex

[0105] First, prepare the reaction system buffer F (20mM HEPES (pH7.2), 50mM NaCl), and add the helicase Dda and the linker Ad3 and Ad3-no pho to the buffer system at a ratio of 10:1, respectively. Place the reaction in a metal bath at 28°C for 1 hour, and wait for the helicase Dda and the Ad3 linker to be fully mixed to obtain a mixed solution.

[0106] Then, 0.3 mM of the oxidant N,N,N',N'-tetramethylazodicarbonamide (TMAD) and 5 mM of EDTA were added to the mixture of helicase Dda and Ad3 linker, and the mixture was placed in a metal bath at 28°C for 90 minutes to allow the pin domain and tower domain of helicase Dda to be stably cross-linked (the purpose is to fix the linker in the "arch" structure cavity it forms, facilitating subsequent sequencing work) to obtain a cross-linking solution.

[0107] Finally, an equal volume of unwinding buffer G (50 mM HEPES (pH 8.0), 1000 mM KCl, 4 mM ATP, 20 mM MgCl2) was added to the cross-linked helicase Dda and Ad3 adapter complex system, and the mixture was placed in a metal bath at 30°C for 90 minutes to allow the complete removal of non-target helicase Dda and Ad3 adapter complexes (e.g., helicase bound to the bottom strand of the adapter).

[0108] 2. Purification of crude Dda-Ad3 linker complex

[0109] The buffer solution used in this step is prepared as follows:

[0110] Buffer H: 50mM Tris (pH7.5), 2.5M NaCl;;

[0111] Buffer I: 50mM Tris (pH7.5), 2.5M NaCl, 24% PEG8000, 0.05% TWEEN20;

[0112] Buffer J: 50mM Tris (pH7.5), 2.5M NaCl, 20% PEG8000;

[0113] Buffer K: 50mM Tris (pH7.5), 20mM NaCl.

[0114] Magnetic beads (VAHTS™ DNA Clean Beads #N411-03) were used to purify the reaction system to remove free proteins, crosslinkers, and nonspecifically bound non-target complexes. The specific steps are as follows: First, remove the magnetic beads from the refrigerator, vortex to mix thoroughly, and then bring them to room temperature. Wash the beads with Buffer H (magnetic bead wash buffer) to replace the buffer in the beads with the appropriate high-salt buffer pH used in the purification process. Then, equilibrate the beads with Buffer I (magnetic bead equilibration buffer). Then, mix the equilibrated beads with the optimized helicase Dda and linker Ad3 complex constructed above at twice the initial volume. Place the beads in a low-binding centrifuge tube and allow the mixture to bind on a rotary shaker at room temperature for 1 hour. The tube was then placed on a magnetic stand for 10 minutes. Once the beads were completely attracted to the stand and the solution was clear, carefully remove the supernatant. The mixed solution of magnetic beads and complexes is then washed with Buffer J (magnetic bead equilibration buffer). After each thorough mixing, wait until the magnetic beads are completely adsorbed to the side of the magnetic rack and the solution becomes completely clear before carefully removing the supernatant. This is to remove free proteins, cross-linkers, nonspecifically bound non-target complexes, and excess surfactants from the solution. Finally, after the solution in the system is fully removed, the centrifuge tube is placed on the magnetic rack and allowed to stand. After the surface of the magnetic beads is dry, Buffer K (low salt elution buffer) is added, the magnetic beads are resuspended, and the solution is allowed to stand at room temperature for 5 minutes to purify and elute the target complex. 2 μL of the purified product is quantified using the Qubit dsDNA HS kit (Thermofisher, Q32854), and the eluted product is collected to obtain the complex with a 5' phosphorylated linker.

[0115] The preparation methods of Ad3 and Ad3-no pho were the same as those in Example 3. A complex without a phosphorylated linker at the 5' end was prepared in the same manner.

[0116] Example 4 Sequencing library construction with exonuclease step:

[0117] The complete process of the embodiment of the present invention (including: initial nucleic acid molecule fragmentation (optional), end repair and A addition, adapter ligation, nuclease treatment, and anchor sequence annealing) is shown in Figure 2. Sequencing libraries containing Ad3 adapters and Ad3-no pho adapters were constructed respectively. The specific steps are as follows:

[0118] 1. DNA shearing (optional)

[0119] If the initial genome input is less than 12 μg, DNA fragmentation can be performed to increase library capture efficiency during sequencing. We recommend using the Covaris g-TUBE (Covaris, 520079) for fragmentation. For details on the fragmentation system and procedures, refer to the g-TUBE manual.

[0120] 2. End repair plus "A"

[0121] 2.1 In a 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051), calculate the volume of sample required to produce 12 μg of total DNA based on the sample concentration. Add Nuclease-Free Water (Thermofisher, AM9932) to normalize the sample volume to 288 μL.

[0122] 2.2 Prepare the required amount of End Repair plus "A" reaction mixture in a centrifuge tube according to the ratios in Table 1. This step should be prepared on ice. Vortex the prepared End Repair plus "A" reaction mixture three times for 3 seconds each, then centrifuge briefly to collect the reaction mixture at the bottom of the tube.

[0123] Table 1: End Repair Plus "A" Reaction Mix

[0124] Components: Single reaction volume: FFPE DNA repair mix (NEB, M6630LVIAL) 12 μL FFPE DNA repair buffer (E6622AAVIAL) 21 μL End repair enzyme mix (NEB, E6051AAVIAL) 18 μL End repair reaction buffer (NEB, E6052AAVIAL) 21 μL Total volume: 72 μL

[0125] 2.3 Use a pipette to draw 72 μL of the prepared end-repair reaction mixture plus "A" into the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) prepared in step 2.1. Gently pipette with a flared pipette tip to mix, or gently tap the tube wall to mix. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0126] 2.4 Use a flared pipette tip to dispense 360 ​​μL of the end-repair reaction solution into 6 PCR tubes, with 60 μL in each tube. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0127] 2.5 Place the PCR tube in 2.4 into a PCR instrument and perform the reaction according to the conditions in Table 2.

[0128] Table 2: End Repair Plus "A" Reaction Conditions

[0129] Temperature time

[0130] Hot cover (105℃) ON 20℃ 10min 65℃ 10min 4℃ Hold

[0131] 2.6 Centrifuge briefly to collect the reaction solution at the bottom of the tube. Combine the six tubes of end-repaired samples with "A" and transfer them to a clean 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051).

[0132] 3. End repair plus "A" product purification

[0133] 3.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4°C refrigerator 30 min in advance, shake and mix thoroughly, then place at room temperature. Shake and mix thoroughly before use.

[0134] 3.2 Pipette 360 ​​μL of magnetic beads into the sample in 2.6. Mix thoroughly by gently tapping the tube wall, or gently pipette at least 6 times with a flared pipette tip until thoroughly mixed. The last time, ensure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

[0135] 3.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0136] 3.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid becomes clear. Carefully aspirate the supernatant with a pipette and discard it.

[0137] 3.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack and add 750 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0138] 3.6 Repeat step 3.5. Remove the centrifuge tube from the magnetic rack and centrifuge briefly. After separation on the magnetic rack, use a small-range pipette to remove the remaining liquid at the bottom of the tube.

[0139] 3.7 Place the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, open the tube cap, and dry at room temperature until the surface of the magnetic beads is free of reflections and cracks.

[0140] 3.8 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic stand and add 392 μL of Nuclease-Free Water (Thermofisher, AM9932) to elute the DNA. Gently flick the tube to mix. Centrifuge for 3 seconds and collect the liquid at the bottom of the tube.

[0141] 3.9 Incubate at room temperature for 5 minutes.

[0142] 3.10 Briefly centrifuge a DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) and place on a magnetic rack. Let stand for 2–5 minutes until the liquid clears. Transfer 390 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. The Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) is recommended for determining the concentration of the purified end-repaired "A" product.

[0143] 4. Connector connection

[0144] 4.1 Set the metal bath temperature to 25°C for preheating.

[0145] 4.2 Remove the adapters (Ad3, Ad3-no pho) and T4 DNA ligase (NEB, E6057AVIAL) prepared in Example 3 from the -20°C freezer, gently tap the tube to mix, centrifuge briefly, and place on ice. Thaw Quick Ligation Reaction Buffer (NEB, E6058AVIAL), pipette to mix, centrifuge briefly, and place on ice. Prepare the ligation reaction mixture according to Table 3.

[0146] Table 3: Ligation Reaction Mix

[0147] Components: Single reaction volume: Rapid Ligation Reaction Buffer (NEB, E6058AVIAL) 120 μL T4 DNA Ligase (NEB, E6057AVIAL) 60 μL Total volume: 180 μL

[0148] 4.3 Add 30 μL of the Ad3 / Ad3-no pho linker prepared in Example 1 to a 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) containing 390 μL of the purified end-repaired "A" product from 3.10. Gently pipette up and down six times with a wide-mouth pipette tip to mix. Centrifuge briefly to collect the reaction mixture at the bottom of the tube.

[0149] 4.4 Use a pipette to slowly draw 180 μL of the prepared adapter ligation reaction mixture into the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) prepared in step 4.3. Gently pipette and mix six times with a flared pipette tip. Centrifuge briefly to collect the reaction mixture at the bottom of the tube.

[0150] 4.5 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.4 into the metal bath preheated at 25°C in step 4.1 to carry out the ligation reaction. Set the timer to 30 min.

[0151] 4.6 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

[0152] 5. Purification of Ligation Products

[0153] 5.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4°C refrigerator 30 minutes in advance, shake and mix thoroughly, then bring to room temperature. Shake and mix thoroughly before use.

[0154] 5.2 Pipette 240 μL of magnetic beads into the sample in 4.6. Mix thoroughly by gently tapping the tube wall, or gently pipette at least 6 times with a flared pipette tip until thoroughly mixed. Ensure that all liquid and magnetic beads in the pipette tip are pumped into the tube with the last stroke.

[0155] 5.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0156] 5.4 Centrifuge a DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid becomes clear. Carefully aspirate the supernatant with a pipette and discard it.

[0157] 5.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack. Add 900 μL of Wash Buffer 1 (for short fragments) or Wash Buffer 2 (for long fragments). Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack and gently tap the tube to mix the magnetic beads. After mixing, return the tube to the magnetic rack and let it sit for 2-5 minutes until all the magnetic beads are attached to the wall. Carefully aspirate and discard the supernatant.

[0158] 5.6 Repeat step 5.5. Remove the centrifuge tube from the magnetic rack and centrifuge briefly. After separation on the magnetic rack, use a small-scale pipette to remove the remaining liquid at the bottom of the tube.

[0159] 5.7 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic stand and add 68 μL of elution buffer to elute the DNA. Gently flick the tube to mix. Centrifuge for 3 seconds and collect the liquid at the bottom of the tube.

[0160] 5.8 Incubate at room temperature for 10 minutes. If the library insert is long, incubate at 37°C for 10 minutes.

[0161] 5.9 Briefly centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) and place on a magnetic rack. Let stand for 2–5 minutes until the liquid clears. Transfer 66 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. The Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) is recommended for determining the concentration of the purified ligation product.

[0162] 6. Enzyme Digestion

[0163] 6.1 Set the metal bath temperature to 37°C for preheating.

[0164] 6.2 Combine T7 Exonuclease (T7 exonuclease; NEB, M0263LVIAL) and NE Buffer TMRemove 4 (NEB, B7004SVIAL) from the kit, vortex three times for 3 seconds each, centrifuge briefly, and place on ice. Prepare the enzyme digestion reaction mixture according to Table 4.

[0165] Table 4: Enzyme digestion reaction mixture

[0166] Components: Single reaction volume: NE Buffer 4 (NEB, B7004SVIAL) 7.5 μL T7 Exonuclease (NEB, M0263LVIAL) 1.5 μL Total volume: 9 μL

[0167] 6.3 Add 9 μL of the enzyme digestion reaction mixture to the 1.5 mL DNA LoBind Microcentrifuge Tube containing 66 μL of the purified ligation product in 5.9. Gently pipette up and down six times with a wide-mouth pipette tip to mix thoroughly. Centrifuge briefly to collect the reaction mixture at the bottom of the tube.

[0168] 6.4 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from step 6.3 in the 37°C preheated metal bath from step 6.1 to perform the ligation reaction. Set the timer to 5 minutes.

[0169] 6.5 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

[0170] 7. Purification of Enzymatic Digestion Products

[0171] 7.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4°C refrigerator 30 minutes in advance, shake and mix thoroughly, then bring to room temperature. Shake and mix thoroughly before use.

[0172] 7.2 Pipette 75 μL of magnetic beads into the sample in 6.5. Mix thoroughly by gently tapping the tube wall, or gently pipette at least 6 times with a flared pipette tip until thoroughly mixed. The last time, ensure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

[0173] 7.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0174] 7.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid becomes clear. Carefully aspirate the supernatant with a pipette and discard it.

[0175] 7.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack. Add 200 μL of Wash Buffer 1 (for short fragments). Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack and gently tap the tube to mix the magnetic beads. After mixing, return the tube to the magnetic rack and let it sit for 2-5 minutes until all the beads are attached to the wall. Carefully aspirate and discard the supernatant.

[0176] 7.6 Repeat step 7.5. Aspirate as much liquid as possible from the tube. If a small amount remains on the tube wall, centrifuge the tube briefly. After separation on a magnetic stand, use a small-scale pipette to aspirate the liquid at the bottom of the tube.

[0177] 7.7 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic stand and add 62 μL of elution buffer to elute the DNA. Gently flick the tube to mix. Centrifuge for 3 seconds and collect the liquid at the bottom of the tube.

[0178] 7.8 Incubate at room temperature for 10 minutes. If the library is too long, incubate at 37°C.

[0179] 7.9 Briefly centrifuge a DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) and place on a magnetic rack. Let stand for 2–5 minutes until the liquid becomes clear. Transfer 60 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. The Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) is recommended for concentration determination of the purified enzyme digestion product.

[0180] 7.10 The entire library construction process is now completed and the product can be stored in a 4-degree refrigerator for 72 hours.

[0181] 7.11 Plasmid digestion bands were used as quality control samples for the above library construction process. The comparison results before and after digestion are shown in Figure 5. The results show that three types of molecules are generated after ligation: the original template without adapters, the library with single-ended adapters, and the library with double-ended adapters. After exonuclease treatment, the number of products without adapters and those with single-ended adapters was significantly reduced. Moreover, the number of products with double-ended adapters did not decrease significantly with increasing digestion time, indicating that the target double-ended adapter ligation products can be effectively enriched after exonuclease treatment.

[0182] Example 5: Nanopore sequencing

[0183] The library to be tested prepared in Example 4 was subjected to nanopore sequencing to verify the advantages of the sequencing library constructed in the present application in nanopore sequencing, namely, on the one hand, by introducing exonucleases to degrade the test fragments that have not been completely connected, the products of incomplete connector connection, and the unconnected connectors, the number of abnormal sequencing is reduced, and the sequencing accuracy and sequencing throughput are improved. On the other hand, by using a 5'-end non-phosphorylated connector and treating the connected product with exonucleases, the connector itself is prevented from connecting to form a dimer, and the free connector that is not connected to the two ends of the inserted fragment to be tested is digested into a single chain, thereby reducing the residual connector sequence that may be sequenced or reducing the time it occupies the sequencing channel, thereby improving the sequencing throughput. At the same time, since the chain without phosphorylation modification at the 5' end will be further degraded by the exonuclease to expose the anchor sequence binding position, and the anchor sequence itself will not be covalently bound to the library to be tested, after the motor protein passes through the anchor sequence binding position, the anchor sequence will be stripped from the sequencing library itself, thereby realizing the reuse of the anchor sequence and improving the library capture efficiency.

[0184] 1. With reference to the single-channel electrophysiological detection system in Geng Jia and Guo Peixuan (“Application of phage phi29 DNA packaging motor phospholipid membrane chimera in single molecule detection and nanomedicine”. Life Science, 2011, 23(11):1114-1129), a nanopore detection platform based on the patch clamp platform was constructed, and porin (Sigma-Aldrich, H9395-5mg) was inserted into the phospholipid bilayer membrane to form a single-channel nanopore.

[0185] 2. Sequencing the two libraries constructed in Example 4, it can be seen that the single-channel sequencing reads per unit time for the 5' unphosphorylated adapter are significantly higher than the reads for the 5' phosphorylated adapter (Figure 6). This is because the DNA strand without phosphorylation modification at the 5' end is degraded by the nuclease exonuclease, exposing the anchor sequence binding site. The anchor sequence itself does not covalently bind to the library to be tested. After the motor protein passes through the anchor sequence binding site, the anchor sequence is stripped from the sequencing library itself, thereby achieving reuse of the anchor sequence, improving the library capture efficiency, and resulting in an increase in sequencing throughput per unit time.

[0186] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0187] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0188] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for establishing a sequencing library, characterized in that: include: The sample to be tested connected with the adapter is digested under the action of nuclease to obtain the sequencing library; wherein the 5' end of the adapter is not phosphorylated, and the 3' end of the adapter is connected to the 5' end of the sample to be tested.

2. The method according to claim 1, characterized in that: The sample to be tested connected with the adapter has a gap, and the gap is located between the 5' end of the adapter and the 3' end of the sample to be tested.

3. The method according to claim 1, characterized in that The method further comprises performing a dehelication treatment on the digestion product; Optionally, the unwinding process is performed under the action of a helicase.

4. The method according to claim 3, characterized in that The helicase moves along the DNA in a direction from the 5' end to the 3' end, and the exonuclease is a 5'->3' exonuclease; Optionally, the 5'->3' exonuclease recognizes double-stranded DNA, and the exonuclease comprises at least one selected from T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, Exonuclease VIII (truncated); Preferably, the exonuclease is T7 exonuclease.

5. The method according to claim 3, characterized in that: The helicase moves along the DNA in a direction from the 3' end to the 5' end, and the exonuclease is a 3'->5' exonuclease; Optionally, the 3'->5' exonuclease recognizes double-stranded DNA, and the exonuclease comprises at least one selected from Exonuclease III, Exonuclease IX, and Exonuclease X.

6. The method according to claim 1, characterized in that The exonuclease recognizes single-stranded DNA.

7. The method according to claim 1, characterized in that The method further comprises performing an anti-degradation modification treatment on the 5' end and / or the 3' end of the sample to be tested connected with the adapter; Optionally, the anti-degradation modification includes at least one selected from phosphate modification, 2'-OH modification (RNA base), 2'-F modification, LNA locked nucleotide modification and PNA peptide nucleic acid modification.

8. The method according to claim 1, characterized in that The digestion treatment is carried out at 37° C. and at a ratio of 44:1 between the ligation product and T7 exonuclease for 4 to 6 minutes.

9. The method according to claim 1, characterized in that: The connector is a Y-type connector or a non-Y-type connector; Optionally, the non-Y-shaped connector has at least one of the following structures: Complete complementary double strands, complementary double strands-non-complementary single strands-complementary double strands, 5' protruding single strands-complementary double strands, 3' protruding single strands-complementary double strands.

10. The method according to claim 1, characterized in that The method further comprises purifying the digestion product; optionally, the purification process uses Ampure XP magnetic beads for purification.

11. The method according to claim 1, characterized in that: The sample to be tested connected with the connector is obtained in the following manner: The samples to be tested are subjected to end repair and A addition treatment; The product treated with A is subjected to a connector connection treatment to obtain the sample to be tested connected with a connector; Optionally, after the end repair and A addition treatment and before the ligation treatment, the process further comprises performing a first purification treatment on the A addition treatment product; Optionally, after the ligation treatment, the method further comprises subjecting the ligation product to a second purification treatment.

12. The method according to claim 1, characterized in that The sample to be tested is a DNA sample.

13. The method according to claim 1, characterized in that Before performing end repair and A addition treatment on the sample to be tested, the sample to be tested is further subjected to fragmentation treatment.

14. A sequencing library, characterized in that: The sequencing library is obtained by the method according to any one of claims 1 to 13.

15. A sequencing method, characterized in that: include: Mixing the sequencing library of claim 14 with an anchor sequence, wherein at least a portion of the anchor sequence is complementary to at least a portion of one strand of the adapter; The mixed processing product is subjected to sequencing processing to obtain the sequence of the sample to be tested; Optionally, the sequencing is performed on a nanopore sequencing platform.

16. A kit for constructing a sequencing library or sequencing, characterized in that: The method comprises: a reagent, wherein the reagent comprises a nuclease exonuclease, and is used for digesting a sample to be tested connected with a connector; Optionally, further comprising a first reagent, wherein the first reagent is suitable for fragmenting the sample to be tested; Optionally, the method further comprises a second reagent, wherein the second reagent is suitable for performing end repair and A addition treatment on the sample to be tested; Optionally, a third reagent is further included, and the third reagent is suitable for performing a connector connection treatment on the sample to be tested.

17. The kit according to claim 16, characterized in that The kit further comprises a linker, wherein the 5' end of the linker is not phosphorylated.

18. The kit according to claim 16, characterized in that The exonuclease includes at least one of T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X.

19. The kit according to claim 16, characterized in that The kit further comprises an anchor sequence, at least a portion of which complementarily pairs with at least a portion of one strand of the adaptor.

Images