Method for reducing free linkers in sequencing library

Abstract

The invention provides a method for constructing a sequencing library, the sequencing library, a sequencing method and a kit for constructing the sequencing library. The method for constructing 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 a target sequencing library.

Description

Methods for reducing free adapters in sequencing libraries Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a method for constructing a sequencing library and its application, and more particularly, to a method for constructing a sequencing library, a sequencing library, a method for sequencing a target nucleic acid molecule, and a kit. 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 an urgent need in the art to develop a method for removing free adapters in sequencing libraries.

[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] To avoid the rate-limiting protein denaturation problem caused by the use of alcohol detergents during column purification, the inventors discovered that a nuclease exonuclease can be added during library construction to remove residual linkers in the library. During the purification process, no alcohol detergent is used, and only magnetic beads or column purification is used. This avoids the detection of non-target fragments during sequencing, improves sequencing accuracy, 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 the sample to be tested connected with a connector under the action of a nuclease to obtain the sequencing library. According to the method of an embodiment of the present invention, by introducing a nuclease, the fragment to be tested that has not been connected at all, the single-stranded free connector that is not connected to the two ends of the inserted fragment to be tested, and the product with incomplete connector connection (referring to a product with only one end connected to a connector and the other end not connected to a connector) are degraded, thereby reducing the time that the sequencing channel time is occupied by the non-desired library, thereby improving the sequencing throughput and increasing the sequencing accuracy.

[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 method includes exonuclease digestion of unligated test fragments, free adapters not connected to both ends of the test insert, and products of incomplete adapter ligation to obtain digestion products.

[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 exonuclease recognizes double-stranded DNA and is a 5'->3' exonuclease, and the exonuclease includes at least one selected from T7 exonuclease, Lambda exonuclease, T5 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 may further include 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 hydrolyze 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 strand, such as the 5' end of the sequence to be tested to which the Y-type adapter is connected, may 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 discovered that the sequencing library obtained using this method significantly reduces the number of unconnected test fragments, free adapters not connected to both ends of the test insert, and products resulting from incomplete adapter ligation. Furthermore, the method is simple to operate, achieves significantly higher sequencing accuracy, good reproducibility, low cost, and high 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 sequencing the aforementioned sequencing library to obtain the sequence of the sample to be tested. The inventors have discovered that this method can efficiently determine the sequence information of nucleic acid samples with high sensitivity, high accuracy, good reproducibility, and high sequencing throughput.

[0040] According to a specific embodiment of the present invention, the sequencing is performed on a nanopore sequencing platform. When the sequencing is performed on a nanopore sequencing platform, the sequencing adapter is generally coupled to a rate-control protein necessary for sequencing. Therefore, during magnetic bead or column purification after ligation, alcohol-containing cleaning reagents cannot be used. When the sequencing library constructed according to the first aspect of the present invention is used, unligated test fragments, free adapters not connected to the ends of the test insert, and products of incomplete adapter ligation are degraded during the sequencing library purification, thereby avoiding the detection of non-target fragments during the sequencing process and significantly improving sequencing accuracy.

[0041] In a fourth aspect of the present invention, the present invention further provides a kit for constructing a sequencing library. According to an embodiment of the present invention, the kit includes a reagent containing a nuclease exonuclease for digesting a sample to be tested to which a connector is connected. The inventors have found that by using the kit of the present invention, in combination with the above-mentioned method for establishing a sequencing library, a sequencing library, and a sequencing method, a sequencing library can be effectively obtained and effectively applied to a high-throughput sequencing platform, thereby effectively determining the nucleic acid sequence information of the nucleic acid sample, and the obtained information has high accuracy and high sequencing throughput.

[0042] According to an embodiment of the present invention, the kit further includes at least one of the following additional technical features:

[0043] 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.

[0044] 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.

[0045] 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.

[0046] According to an embodiment of the present invention, the kit further comprises a linker, wherein the linker carries a 5' terminal phosphate group.

[0047] 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.

[0048] 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

[0049] 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:

[0050] FIG1 is a diagram illustrating the Pacbio circularization consensus sequencing principle according to an embodiment of the present invention;

[0051] FIG2 is a flowchart of exonuclease-free nanopore library construction according to an embodiment of the present invention;

[0052] FIG3 shows a library construction process including an exonuclease step according to an embodiment of the present invention (sequencing direction is 5'->3');

[0053] FIG4 shows a library construction process including an exonuclease step according to an embodiment of the present invention (sequencing direction is 3'->5');

[0054] FIG5 is a protein control result after purification according to an embodiment of the present invention;

[0055] FIG6 is a comparison of library bands before and after enzyme digestion according to an embodiment of the present invention;

[0056] FIG7 is a comparison of changes in electrical signals of libraries with and without enzyme digestion according to an embodiment of the present invention;

[0057] FIG8 shows statistics on the proportion of free adapters in original library sequencing and enzyme-digested library sequencing 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, the present invention provides a method for establishing a sequencing library. According to an embodiment of the present invention, referring to Figures 3 and 4, the method comprises the following steps:

[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 embodiments of the present invention, exonuclease can select at least one of T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X. In the 7th step, the sample processed by exonuclease is subjected to product purification process to obtain a preliminary library sample. In the 8th step, the preliminary library sample obtained is incubated with helicase Dda albumen altogether, and finally obtains the library that can be used for sequencing. According to embodiments of the present invention, the helicase is from 5' to 3' (Fig. 3) or 3' to 5' (Fig. 4) along the direction of movement of DNA.

[0064] According to an embodiment of the present invention, the helicase and T7 exonuclease can be added simultaneously.

[0065] 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.

[0066] Acquisition of sequencing libraries

[0067] The library of the sample to be tested is obtained by the above-mentioned method for establishing a sequencing library.

[0068] Sequencing methods

[0069] According to a specific embodiment of the present invention, a library sample to be tested is sequenced on a nanopore sequencing platform. Sequencing is achieved by electrophoresis, which drives individual molecules through the nanopore one by one. Due to the extremely small diameter of the nanopore, only a single nucleic acid polymer can pass through. Because individual ATCG bases have different charge properties, the difference in the electrical signal can be used to detect the type of base that passed through, thereby achieving sequencing.

[0070] Motor proteins

[0071] 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.

[0072] Primers

[0073] 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.

[0074] iSpC3

[0075] iSpC3 is a three-hydrocarbon carbon chain that is commonly used as a spacer in oligonucleotide chains.

[0076] iSp18

[0077] iSp18 is an 18-atom-long hexaethylene glycol chain that is commonly used as a spacer in oligonucleotide chains.

[0078] 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.

[0079] Example 1: Preparation of Ad3 linker sequence

[0080] In this example, the Ad3 linker sequence was prepared by annealing chemically synthesized SEQ ID NO. 1 and SEQ ID NO. 2.

[0081] 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.

[0082] 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3' (X=iSpC3; Y=iSp18; SEQ ID NO. 1)

[0083] 5'pho-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3'(SEQ ID NO.2)

[0084] 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 were mixed together and thoroughly vortexed. The mixture was heated to 70°C in a thermal cycler and incubated for 10 minutes. The temperature was then cooled to 25°C at a rate of 0.1°C / s and incubated for another half an hour. This yielded a 10 μM annealed linker solution. The linker product was named Ad3 and stored in a -20°C refrigerator.

[0085] Example 2. Cloning, expression and purification of helicase Dda

[0086] In this example, helicase Dda (amino acid sequence shown in SEQ ID NO. 4) was prepared by recombinant expression in Escherichia coli, and the helicase was used as a motor protein.

[0087] 1. Order the full-length Dda cDNA sequence from Sangon Biotechnology (nucleotide sequence shown in SEQ ID NO. 3), ligate it into the pET.28a(+) plasmid, and digest the vector with double enzyme digestion 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.

[0088] 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.

[0089] 3. Prepare five buffer solutions according to the following formula:

[0090] Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

[0091] Buffer B: 20 ​​mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0092] Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

[0093] Buffer D: 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;

[0094] Buffer E: 20 mM Tris-HCl pH 7.5, 100 mM NaCl.

[0095] 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 5.

[0096]

[0097]

[0098] Example 3 Sequencing library construction with exonuclease step:

[0099] The complete process of the embodiment of the present invention is shown in Figure 3. The sequencing direction is selected as 5'->3'. The main steps of constructing the sequencing library include: initial nucleic acid molecule fragmentation (optional), end repair and A addition, adapter ligation, nuclease treatment, and anchor sequence annealing.

[0100] 1. DNA shearing (optional)

[0101] 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.

[0102] 2. End repair plus "A"

[0103] 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.

[0104] 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.

[0105] Table 1: End Repair Plus "A" Reaction Mix

[0106] 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

[0107] 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.

[0108] 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.

[0109] 2.5 Place the PCR tube in 2.4 into a PCR instrument and perform the reaction according to the conditions in Table 2.

[0110] Table 2: End Repair Plus "A" Reaction Conditions

[0111] Temperature Time Hot Cover (105℃) ON 20℃ 10min 65℃ 10min 4℃ Hold

[0112] 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).

[0113] 3. End repair plus "A" product purification

[0114] 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.

[0115] 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.

[0116] 3.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 3.9 Incubate at room temperature for 5 minutes.

[0123] 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.

[0124] 4. Connector connection

[0125] 4.1 Set the metal bath temperature to 25°C for preheating.

[0126] 4.2 Remove the adapter (Ad3) and T4 DNA ligase (NEB, E6057AVIAL) from the -20°C freezer, gently tap the tube to mix, centrifuge briefly, and place on ice. Thaw the Quick Ligation Reaction Buffer (NEB, E6058AVIAL), pipette to mix, centrifuge briefly, and place on ice. Prepare the ligation reaction mix according to Table 3.

[0127] Table 3: Ligation Reaction Mix

[0128] Components: Single reaction volume: Rapid Ligation Reaction Buffer (NEB, E6058AVIAL) 120 μL T4 DNA Ligase (NEB, E6057AVIAL) 60 μL Total volume: 180 μL

[0129] 4.3 Add 30 μL of the Ad3 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. Centrifuge briefly to collect the reaction mixture at the bottom of the tube.

[0130] 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.

[0131] 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.

[0132] 4.6 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

[0133] 5. Purification of Ligation Products

[0134] 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.

[0135] 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.

[0136] 5.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 5.8 Incubate at room temperature for 10 minutes. If the library insert is long, incubate at 37°C for 10 minutes.

[0142] 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.

[0143] 6. Enzyme Digestion

[0144] 6.1 Set the metal bath temperature to 37°C for preheating.

[0145] 6.2 Combine T7 Exonuclease (NEB, M0263LVIAL) and NE Buffer TM Remove 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.

[0146] Table 4: Enzyme digestion reaction mixture

[0147] Component single reaction volume NE Buffer TM 4 (NEB, B7004SVIAL) 7.5μL T7 Exonuclease (NEB, M0263LVIAL) 1.5μL Total volume 9μL

[0148] 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.

[0149] 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.

[0150] 6.5 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

[0151] 7. Purification of Enzymatic Digestion Products

[0152] 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.

[0153] 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.

[0154] 7.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 7.8 Incubate at room temperature for 10 minutes. If the library is too long, incubate at 37°C.

[0160] 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.

[0161] 7.10 The entire library construction process is now completed and the product can be stored in a 4-degree refrigerator for 72 hours.

[0162] 7.11 Using a plasmid digestion product as a quality control, the library construction process described above was repeated. A comparison of the pre- and post-digestion results revealed three types of molecules after ligation: the original template without an adapter, the library with a single-ended adapter, and the library with a double-ended adapter. After exonuclease treatment, the number of products without an adapter and with a single-ended adapter decreased significantly. Furthermore, the number of products with double-ended adapters did not decrease significantly with increasing digestion time, indicating that this enzymatic treatment effectively enriched the target double-ended adapter-ligated products (Figure 6).

[0163] Example 4 Sequencing library construction without exonuclease step

[0164] The complete process of the embodiment of the present invention is shown in FIG2 .

[0165] 1. DNA shearing (optional)

[0166] 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.

[0167] 2. End repair plus "A"

[0168] 89122.1 was added to a 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The volume of sample required to obtain 12 μg of total DNA was calculated based on the sample concentration. Nuclease-free water (Thermofisher, AM9932) was added to normalize the sample volume to 288 μL.

[0169] 2.2 Prepare the required amount of End Repair plus "A" reaction mixture in a centrifuge tube according to the ratios in Table 5. 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.

[0170] Table 5: End Repair Plus "A" Reaction Mix

[0171] 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

[0172] 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.

[0173] 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.

[0174] 2.5 Place the PCR tube in 2.4 into a PCR instrument and perform the reaction according to the conditions in Table 6.

[0175] Table 6: End Repair Plus "A" Reaction Conditions

[0176] Temperature Time Hot Cover (105℃) ON 20℃ 10min 65℃ 10min 4℃ Hold

[0177] 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).

[0178] 3. End repair plus "A" product purification

[0179] 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.

[0180] 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.

[0181] 3.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0182] 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.

[0183] 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.

[0184] 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 centrifuge tube.

[0185] 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.

[0186] 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.

[0187] 3.9 Incubate at room temperature for 5 minutes.

[0188] 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.

[0189] 4. Connector connection

[0190] 4.1 Set the metal bath to 25°C for preheating.

[0191] 4.2 Remove the adapter (Ad3) and T4 DNA ligase (NEB, E6057AVIAL) from the -20°C freezer, gently tap the tube to mix, centrifuge briefly, and place on ice. Thaw the Quick Ligation Reaction Buffer (NEB, E6058AVIAL), pipette to mix, centrifuge briefly, and place on ice. Prepare the ligation reaction mix according to Table 7.

[0192] Table 7: Ligation Reaction Mix

[0193] Components: Single reaction volume: Rapid Ligation Reaction Buffer (NEB, E6058AVIAL) 120 μL T4 DNA Ligase (NEB, E6057AVIAL) 60 μL Total volume: 180 μL

[0194] 4.3 Add 30 μL of the Ad3 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.

[0195] 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.

[0196] 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.

[0197] 4.6 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

[0198] 5. Purification of Ligation Products

[0199] 5.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4°C refrigerator 30 min in advance, shake and mix thoroughly, then bring to room temperature. Shake and mix thoroughly before use.

[0200] 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.

[0201] 5.3 Incubate at room temperature on a rotary mixer for 5 minutes.

[0202] 5.4 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 5.8 Incubate at room temperature for 10 minutes. If the library insert is long, incubate at 37°C for 10 minutes.

[0207] 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.

[0208] Example 5: Sequencing library preparation

[0209] In this example, the sequencing libraries obtained in Example 3 and Example 4 were incubated with the helicase Dda protein prepared in Example 2, respectively, to prepare the libraries ultimately used for sequencing.

[0210] 1. Add 100 mL of 1 M Tris-HCl pH 7.5 buffer and 100 mL of 1 M KCl solution to a volumetric flask and dilute to 1 L with ultrapure water to prepare 2X binding buffer.

[0211] 2. Prepare a mixed solution of helicase Dda (prepared in Example 2) and the library on ice according to Table 8 below, and then incubate at 30°C for one hour.

[0212] Table 8: Motor protein and library binding system

[0213] Reagent volume 2x binding buffer 50 μL helicase Dda 20 μL purified ligation product 20 μL water 10 μL

[0214] 3. Quantify the library using the Qubit DNA HS kit. After clearly marking the concentration, store the product in a 4°C refrigerator until use.

[0215] Example 6: Nanopore sequencing

[0216] This example uses a nanopore detection platform based on a patch clamp platform to perform nanopore sequencing on the target sequencing library prepared in Example 6 to verify the advantages of the sequencing library constructed in this application in nanopore sequencing, namely, the increase in the number of sequenced molecules.

[0217] 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.

[0218] 2. The sequencing library obtained in Example 5 was added to the single-channel system, and the current amplitude changes were detected and recorded using a patch clamp system.

[0219] 3. The current changes per unit time for sequencing the libraries of Example 3 and Example 4 are shown in Figure 7. It can be seen that, per unit time, the number of sequencing sequences in the library after enzyme digestion treatment is significantly increased, the free adapter sequence signal is reduced, the time occupied by the resting potential is shortened, and the sequencing throughput is significantly improved.

[0220] 4. The number of sequencing library reads and free adapter sequences was detected by the adapter signal discrimination algorithm. The comparison of the free adapter ratio between the original library and the enzyme-digested library showed that after enzyme digestion, the measured free adapter ratio decreased from 41.46% in the original library to 8.82%, while the library molecule ratio increased from the original 58.54% to 91.18% (Figure 8).

[0221] 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.

[0222] 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.

[0223] 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, It is 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.

2. The method according to claim 1, It is 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.

3. The method according to claim 2, It is 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, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated); Preferably, the exonuclease is T7 exonuclease.

4. The method according to claim 2, It is 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.

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

6. The method according to claim 1, It is 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.

7. The method according to claim 1, It is 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.

8. The method according to claim 1, It is 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.

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

10. The method according to claim 1, It is 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.

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

12. The method according to claim 1, It is 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.

13. A sequencing library, It is characterized in that The sequencing library is obtained by the method according to any one of claims 1 to 12.

14. A sequencing method, It is characterized in that include: Performing sequencing on the sequencing library of claim 13 to obtain the sequence of the sample to be tested; Optionally, the sequencing is performed on a nanopore sequencing platform.

15. A kit for constructing a sequencing library, It is 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.

16. The kit according to claim 15, It is characterized in that The kit further comprises an adaptor carrying a 5' terminal phosphate group.

17. The kit according to claim 16, It is 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.

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