CN120344725A

CN120344725A - Methods for reducing free adapters in sequencing libraries

Info

Publication number: CN120344725A
Application number: CN202280102106.5A
Authority: CN
Inventors: 王欧; 师虓; 郭斐; 陈俊毅; 季州翔; 章文蔚; 董宇亮
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2025-07-18
Also published as: WO2024138320A1

Abstract

提供了一种构建测序文库的方法、测序文库、测序方法以及构建测序文库的试剂盒。其中，构建测序文库的方法包括：对连接有接头的待测样本在核酸外切酶的作用下消化处理，以便获得目的测序文库。Provided are a method for constructing a sequencing library, a sequencing library, a sequencing method, and a kit for constructing a sequencing library. The method for constructing a sequencing library comprises: digesting a sample to be tested connected with a connector under the action of an exonuclease to obtain a target sequencing library.

Description

Method for reducing free linkers in sequencing libraries

Technical Field

The invention relates to the field of biotechnology, in particular to a method for constructing a sequencing library and application thereof, and more particularly relates to a method for constructing a sequencing library, a method for sequencing target nucleic acid molecules and a kit.

Background

Nucleic acid sequencing has become an indispensable important research tool in the field of life science research. Along with this, genomic technology based on large-scale sequencing data has also played an important role in different research and application directions, such as tracing the cause of complex diseases and dynamic monitoring of the development process thereof, directional breeding of economic crops and animals, research and protection of different biological genetic resources, and the like.

However, a prerequisite for the above studies is how to obtain high-precision, complete nucleic acid sequences using high-throughput parallel sequencing. The sequencing accuracy of the mainstream sequencing-by-synthesis technology is high, the original accuracy of most sequencing bases can reach more than 99.9%, however, the read length is short, and the complete target nucleic acid sequence is difficult to restore by using the short read length sequences, whether re-sequencing alignment or de novo assembly.

Thus, how to obtain long-distance information in nucleic acid sequences has become one of the hot problems in current sequencing technology research. There are two major commercial companies that currently offer long read long sequencing solutions. The first was single molecule real-time sequencing by PacBio Inc. (Pacific Bioscience, abbreviated as Pacbio) in the United states of America and Nanopore sequencing by Oxford Nanopore technology Inc. (Oxford Nanopore, abbreviated as ONT) in the United kingdom. In addition, foreign Quantum SI, genia, domestic companies such as zizano, today, an Xuyuan and the like all enter the technical field and are released by a prototype machine.

With the progress of science and technology, current clinical sample mutation detection is not satisfied to detect only small-range mutation (single nucleotide mutation and small deletion/insertion mutation), some genetic abnormalities caused by large-range structural mutation are gradually analyzed, and as structural mutation detection is sensitive to sequencing read length, long-read long sequencing technology is gradually changed from the scientific and technological service field to the clinical detection field, and the requirements on accuracy and cost control of the sequencing technology are also improved.

In terms of accuracy, the Hi-Fi sequencing method based on circularized consistency sequencing (fig. 1) introduced by Pacbio corporation in 2019 can reach an average sequencing accuracy of 99% or more by repeating 5 times, but the cost is high because the same molecule needs to be repeatedly sequenced for a plurality of times.

To save the cost, the PromethION system recently introduced by oxford nanopore corporation can realize single Gb cost of $ 2-16, which gradually approaches the sequencing cost of synthesis method with wider application in the market. Although the accuracy of the system is still insufficient compared with the cyclized consistency sequencing method of Pacbio, the accuracy of the system can reach 98.4 percent according to the latest disclosed data of oxford nanopore company, and compared with the early nanopore data, the accuracy of the system is obviously improved.

The current nanopore sequencing scheme is low in cost, but the sequencing accuracy still has a certain improvement space. Unlike conventional sequencing by synthesis, nanopore sequencing libraries typically have long inserts, ranging from a few kilobases to millions of bases, and therefore have fewer double-stranded ends than short insert libraries, and are less efficient at adaptor ligation. The nanopore sequencing linker is generally coupled with a speed control protein necessary for sequencing, so that when magnetic beads or columns are purified after connection, a cleaning reagent with alcohols cannot be used, the linker is difficult to remove by a purification step, and the sequencing accuracy is reduced due to non-target fragment detection in the sequencing process.

Thus, there is a strong need in the art to develop a method for removing free linkers from sequencing libraries.

Disclosure of Invention

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

In order to avoid the problem of denaturation of the speed control protein caused by using alcohol cleaning agents in the process of column purification, the inventor discovers that an exonuclease can be added in the process of library establishment to remove residual joints in a library, and in the process of purification, the alcohol cleaning agents are not used, only magnetic beads or columns are used for purification, so that the measurement of non-target fragments in the process of sequencing is avoided, the sequencing accuracy rate can be improved, the sequencing flux can be increased, and high-quality sequencing reads can be obtained in a short time.

Based on the above findings, in a first aspect of the present invention, the present invention proposes a method of creating a sequencing library. According to an embodiment of the invention, the method comprises digestion of the adaptor-ligated test sample with exonuclease to obtain the sequencing library. According to the method provided by the embodiment of the invention, exonuclease is introduced to degrade fragments to be detected which are not connected at all, single-stranded free joints which are not connected at two ends of the insert to be detected and products with incomplete joint connection (namely products with only one end connected with the joint and the other end not connected with the joint), so that the time of a sequencing channel is shortened from being occupied by an unexpected library, the sequencing flux is further improved, and the sequencing accuracy is increased.

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

According to an embodiment of the present invention, the method comprises subjecting a fragment to be detected which is not ligated, a free linker which is not ligated to both ends of an insert to be detected, and a product of incomplete ligation of the linker to exonuclease digestion to obtain a digested product.

According to an embodiment of the present invention, the method further comprises subjecting the obtained digestion treatment product to a de-spinning treatment.

According to an embodiment of the invention, the unwinding treatment is performed under the action of a helicase.

According to an embodiment of the invention, the helicase is 5 'to 3' in the direction of movement of the DNA, and the exonuclease is a 5'- >3' exonuclease.

According to an embodiment of the invention, the exonuclease recognizes double-stranded DNA and the exonuclease is a 5'- >3' exonuclease comprising at least one selected from T7 exonuclease, lambda exonuclease, T5 Exonuclease, exonuclease VI, exonuclease VIII (truncated).

According to an embodiment of the invention, the exonuclease is preferably T7exonuclease.

According to an embodiment of the invention, the helicase is 3 'to 5' in the direction of movement of the DNA, and the exonuclease is a 3'- >5' exonuclease.

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

According to an embodiment of the invention, the exonuclease recognizes single stranded DNA.

According to an embodiment of the present invention, the method for creating a sequencing library may further include performing degradation-preventing modification treatment on the 5 'end and/or the 3' end of the sample to be tested to which the adaptor is attached. For digestion of the ligation product with some exonuclease (e.g.T 7exonuclease, T5exonuclease, lambda exonuclease, exonuclease VI, exonuclease VIII (truncated)) which recognizes the 5' end of the single stranded DNA, some chemical modification of the 5' end of the single strand, e.g.the 5' end of the test sequence to which the Y-adapter is ligated, may be performed in advance to resist degradation by the nuclease.

According to an embodiment of the invention, the degradation preventing modification comprises at least one selected from the group consisting of a phosphate modification, a 2'-OH modification (RNA base), a 2' -F modification, an LNA lock nucleotide modification and a PNA peptide nucleic acid modification.

According to the embodiment of the invention, the digestion treatment is carried out for 4-6 min under the condition that the temperature is 37 ℃ and the ratio of a connection product to T7 exonuclease is 44:1.

According to an embodiment of the invention, the linker is a Y-linker or a non-Y-linker.

According to an embodiment of the invention, the non-Y-type adaptor has at least one of a complete complementary double strand, a complementary double strand-non-complementary single strand-complementary double strand, a 5 'protruding single strand-complementary double strand, and a 3' protruding single strand-complementary double strand.

According to an embodiment of the invention, the method of creating a sequencing library further comprises subjecting the digestion treatment product to a purification treatment.

According to an embodiment of the invention, the purification treatment uses Ampure XP magnetic beads for purification.

According to an embodiment of the present invention, the sample to be tested to which the connector is connected is obtained by:

(1) Performing end repair and A adding treatment (poly-A tail adding) on a sample to be tested;

(2) And (3) carrying out joint connection treatment on the A-added treatment product so as to obtain the sample to be tested connected with the joint.

According to an embodiment of the present invention, after the end repair and the addition a treatment and before the ligation treatment, further comprising subjecting the addition a treated product to a first purification treatment.

According to an embodiment of the present invention, the ligation process further comprises subjecting the ligation product to a second purification process.

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

According to the embodiment of the invention, before the end repair and the A adding treatment are carried out on the sample to be detected, the method further comprises the step of carrying out fragmentation treatment on the sample to be detected.

In a second aspect of the invention, the invention also provides a sequencing library. According to an embodiment of the invention, the sequencing library is a method for establishing a sequencing library according to an embodiment of the invention, and a sequencing library of nucleic acid samples is obtained. The inventor finds that the products of the sequencing library obtained by the method, which are not connected with the fragments to be tested, the free joints which are not connected with the two ends of the insert to be tested and the incomplete joint connection, are obviously reduced, and the method has the advantages of simple operation, obvious high sequencing accuracy, good repeatability, low cost and large sequencing flux.

In a third aspect of the invention, the invention also provides a sequencing method. According to an embodiment of the invention, the method comprises performing a sequencing process on the sequencing library so as to obtain a sequence of a sample to be tested. The inventor discovers that the method can be used for efficiently determining the sequence information of the nucleic acid sample, and has the advantages of high sensitivity, high accuracy, good repeatability and large sequencing flux.

According to a specific embodiment of the invention, the sequencing is performed on a nanopore sequencing platform. When the sequencing is carried out on a nanopore sequencing platform, the sequencing connector is generally coupled with a speed control protein necessary for sequencing, so that a cleaning reagent with alcohols cannot be used when magnetic beads or columns are purified after connection, when the sequencing library constructed according to the first aspect of the invention is utilized, no connected fragments to be tested, free connectors which are not connected to two ends of the inserted fragments to be tested and products with incomplete connector connection are degraded when the sequencing library is purified, the measurement of non-target fragments in the sequencing process is avoided, and the sequencing accuracy is remarkably improved.

In a fourth aspect of the invention, the invention also provides a kit for constructing a sequencing library. According to an embodiment of the invention, the kit comprises reagents containing exonuclease for digestion treatment of the sample to be tested to which the adaptor is attached. The inventor discovers that by utilizing the kit, the method for establishing the sequencing library, the sequencing library and the sequencing method can effectively obtain the sequencing library, can be effectively applied to a high-flux sequencing platform, can effectively determine the nucleic acid sequence information of the nucleic acid sample, and has high accuracy of the obtained information and large sequencing flux.

According to an embodiment of the invention, the kit further comprises at least one of the following additional technical features:

According to an embodiment of the invention, the reagent further comprises a first reagent adapted to fragment the sample to be tested.

According to an embodiment of the invention, the reagent further comprises a second reagent adapted for end-repairing and a-adding the sample to be tested.

According to an embodiment of the invention, the reagent further comprises a third reagent adapted to subject the sample to be tested to a linker ligation process.

According to an embodiment of the invention, the kit further comprises a linker carrying a 5' terminal phosphate group.

According to an embodiment of the invention, the exonuclease comprises at least one of T7 exonuclease,Lambda exonuclease,T5 Exonuclease,Exonuclease VI,Exonuclease VIII(truncated),Exonuclease III,Exonuclease IX,Exonuclease X.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a diagram of Pacbio circularized consistent sequencing principles according to an embodiment of the present invention;

FIG. 2 is a flow chart of the construction of an exonuclease-free nanopore library according to an embodiment of the invention;

FIG. 3 shows a pooling process (sequencing direction 5'- > 3') with an exonuclease step according to an embodiment of the invention;

FIG. 4 shows a pooling process (sequencing direction 3'- > 5') with an exonuclease step according to an embodiment of the invention;

FIG. 5 shows the results of protein control after purification according to an embodiment of the present invention;

FIG. 6 is a comparison of library bands before and after digestion according to an embodiment of the present invention;

FIG. 7 is a comparison of electrical signal changes in a library of enzyme digestion according to an embodiment of the present invention;

FIG. 8 is a plot of free adaptor ratio statistics for raw library sequencing and cut library sequencing according to an embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first", "a second", or a third "may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

Method for creating sequencing library

According to one aspect of the invention, a method of creating a sequencing library is provided. According to an embodiment of the invention, referring to fig. 3 and 4, the method comprises the steps of:

According to the embodiment of the invention, in the first step, the DNA sequence of the genome is selectively interrupted so as to obtain fragmented genome DNA molecules, so that the higher library capturing rate during on-machine sequencing can be improved. In the second step, end repair is performed on the fragmented genomic DNA molecule plus "A". Thirdly, the genomic DNA added with the 'A' is subjected to product purification treatment, so that purer samples are obtained, and the accuracy and the sequencing flux in the sequencing process are increased. And fourthly, connecting the sample obtained after the purification treatment by a connector. According to the embodiments of the present invention, the adaptor ligation refers to the mixing of the adaptor with T4DNA ligase (NEB, E6057 AVIAL) and finally obtaining the adaptor-ligated sample. Fifth, the product purification is performed on the adaptor-ligated samples, and higher purity samples are extremely important for the accuracy of on-machine sequencing and data volume. And sixthly, the purified connection product is digested under the action of exonuclease, and the product which is completely unconnected with the to-be-detected fragment, one chain in free joints which are not connected with two ends of the to-be-detected insertion fragment and is incompletely connected with the joints is degraded, so that the time of a sequencing channel is shortened, the time occupied by an unexpected library is shortened, and the sequencing flux is improved. According to an embodiment of the invention, the exonuclease may be selected to be at least one of T7 exonuclease,Lambda exonuclease,T5 Exonuclease,Exonuclease VI,Exonuclease VIII(truncated),Exonuclease III,Exonuclease IX,Exonuclease X. Seventh, the sample treated by the exonuclease is subjected to product purification treatment, and a preliminary library sample is obtained. And eighth step, incubating the obtained preliminary library sample with the helicase Dda protein to finally obtain a library which can be used for sequencing on a machine. According to an embodiment of the present invention, the helicase is 5 'to 3' or 3 'to 5' in the direction of DNA movement (fig. 3).

According to an embodiment of the invention, the helicase and the T7 exonuclease may be added simultaneously.

According to the embodiment of the invention, the method can be used for preparing the sequencing sample efficiently, and the obtained sequencing library can be effectively applied to a high-throughput sequencing platform, so that the nucleic acid sequence information of the library sample can be effectively determined. In addition, the inventors have surprisingly found that the method for preparing sequencing libraries of the present invention has the advantages of simple process, very easy operation, very easy standardization of operation procedures, easy popularization, low cost, high sensitivity, high accuracy and good repeatability.

Acquisition of sequencing library

A library of samples to be tested is obtained by the method for creating a sequencing library described above.

Sequencing method

According to a specific embodiment of the invention, the library sample to be tested is sequenced on-machine on a nanopore sequencing platform. Sequencing is achieved by driving individual molecules one by one through a nanopore by means of electrophoresis using electrophoresis techniques. Since the diameter of the nanopore is very small, only a single nucleic acid polymer is allowed to pass through. Because the charged properties of the ATCG single bases are different, the types of the bases passing through can be detected through the difference of electric signals, thereby realizing sequencing.

Motor protein

Motor proteins (helicases in example 2 of the present invention) refer to a class of proteins distributed inside or on the surface of cells that are responsible for macroscopic movement of a portion of the material within a cell or of the whole cell.

Primer(s)

The term "primer" as used herein is a generic term for an oligonucleotide that is capable of complementary pairing with a template and is capable of synthesizing a DNA strand complementary to the template under the action of a DNA polymerase. The primer may be natural RNA, DNA, natural nucleotide in any form, or even non-natural nucleotide such as LNA or ZNA.

iSpC3

ISpC3 is a3 hydrocarbon-based carbon chain, often used as a spacer in oligonucleotide chains.

iSp18

Iss 18 is an 18 atom long hexaethyleneglycol chain commonly used as a spacer in oligonucleotide chains.

The invention will be further illustrated with reference to specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 preparation of Ad3 linker sequences

In this example, the Ad3 linker sequences were prepared by annealing the chemically synthesized SEQ ID No.1 and SEQ ID No. 2.

1. The SEQ ID NO.1 and SEQ ID NO.2 primer sequences (organisms) were ordered and the SEQ ID NO.1 and SEQ ID NO.2 primers were dissolved in TE buffer (pH=8) according to the instructions to a final concentration of 100. Mu.M stock. Subsequently, 10. Mu.L of the stock solution was added to 40. Mu.L of TE buffer (pH=8) to dilute the stock solution to a final concentration of 20. Mu.M.

5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3'(X＝iSpC3;Y＝iSp18;SEQ ID NO.1)

5’pho-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3’(SEQ ID NO.2)

Mixing 30 mu L of the working solution of the SEQ ID NO.1 primer and 30 mu L of the working solution of the SEQ ID NO.2 primer obtained by the dilution in the previous step, fully vibrating and uniformly mixing by using a vortex oscillator, heating to 70 ℃ by using a thermal cycler, incubating for 10 minutes, then continuously incubating for half an hour after the temperature is reduced to 25 ℃ according to the cooling rate of 0.1 ℃ per second, obtaining an annealed 10 mu M joint solution, named Ad3 of the joint product, and storing in a refrigerator of-20 ℃.

EXAMPLE 2 cloning, expression and purification of helicase Dda

This example prepares the helicase Dda (amino acid sequence shown in SEQ ID No. 4) by recombinant expression in e.coli, which is used as a motor protein.

1. The full-length cDNA sequence of full-length Dda is ordered from an organism (the nucleotide sequence is shown as SEQ ID NO. 3), and is connected into a PET.28a (+) plasmid, and the vector is cut by double enzyme digestion, wherein the double enzyme digestion sites are Nde1 and Xho1, so that the Dda protein with the 6 XHis tag and thrombin (thrombin) enzyme digestion site at the N end is expressed.

2. The cloned PET.28a (+) -Dda plasmid was transformed into Arcticexpress (DE 3) competent bacteria (Tolo Biotech, 96183-02) or derivatives thereof. Single colonies were picked and transferred to 5mL LB medium containing kanamycin for propagation, and shake culture was performed at 37℃overnight. Then transferring into 1L LB (containing kanamycin), shake culturing at 37deg.C until OD ₆₀₀ is 0.6-0.8, cooling to 16deg.C, adding 500 μm final concentration IPTG to induce Dda expression overnight.

3. Five buffers were prepared according to the following formulation:

Buffer A, 20mM Tris-HCl pH 7.5,250mM NaCl,20mM imidazole;

Buffer B, 20mM Tris-HCl pH 7.5,250mM NaCl,300mM imidazole;

Buffer C, 20mM Tris-HCl pH 7.5,50mM NaCl;

Buffer D, 20mM Tris-HCl pH 7.5,1000mM NaCl;

buffer E, 20mM Tris-HCl pH 7.5,100mM NaCl.

4. The Dda cells expressed in step 2 were collected, resuspended in step 3 buffer a, disrupted with a cell disrupter, and the supernatant was centrifuged. The supernatant was mixed with Ni-NTA filler equilibrated with buffer A in advance and combined for 1h. The packing was collected and washed extensively with buffer a until no contaminating proteins were washed out. The Dda was then eluted by adding buffer B to the packing. And (3) passing the obtained Dda through a desalting column with well balanced buffer solution C, and replacing the buffer solution. An appropriate amount of thrombin (thrombin) (assist organism, 20402ES 05) was then added, and the mixed sample was added to a buffer C-equilibrated ssDNA cellulose (Sigma, D8273-10G) pad, digested at 4 ℃ and combined overnight. The ssDNA cellulose wad was collected, washed 3-4 times with buffer C, and then eluted with buffer D. The protein concentrate after ssDNA cellulose purification was passed through molecular sieve Superdex 200 (Sigma, GE 28-9909-44), buffer E. Collecting target protein, concentrating, and freezing. The concentration of the purified protein was quantified using Nanodrop. The protein was also tested for purity using HPLC and SDS-PAGE electrophoresis, and the results are shown in FIG. 5.

Example 3 construction of sequencing library with exonuclease step:

the complete flow of the embodiment of the invention is shown in figure 3. The sequencing direction was chosen to be 5'- >3'. The main steps of the construction of the sequencing library comprise initial nucleic acid molecule fragmentation (optional), end repair and A addition, linker ligation, nuclease treatment and anchor sequence annealing.

DNA interruption (optional)

If the initial genome input is less than 12. Mu.g, DNA may be fragmented to increase the library capture rate during on-press sequencing. It is recommended to use Covaris g-TUBE (Covaris, 520079) for fragmentation breaks, breaking systems and steps refer to the g-TUBE specification.

2. End repair plus "A"

2.1 In 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051), the total DNA was calculated from the sample concentration to be 12. Mu.g of the sample volume to be added, and the sample volume was homogenized to 288. Mu.L by supplementing nucleic-FREE WATER (Thermofisher, AM 9932).

2.2 According to the ratios in Table 1, the end repair plus "A" reaction mixture was prepared in a centrifuge tube in the amount required for detection. The preparation is carried out on ice, the prepared end repair is added with the A reaction mixed solution for vortex vibration for 3 times, each time for 3s, and the reaction solution is collected to the bottom of the tube by instantaneous centrifugation.

TABLE 1 reaction mixture of end repair and "A

Component (A)	Single reaction volume
FFPE DNA repair mixture (NEB, M6630 LVIAL)	12μL
FFPE DNA repair buffer (E6622 AAVIAL)	21μL
End repair enzyme cocktail (NEB, E6051 AAVIAL)	18μL
Terminal repair reaction buffer (NEB, E6052 AAVIAL)	21μL
Total volume of	72μL

2.3 Sucking 72. Mu.L of the prepared end repair reaction plus A mixed solution by a pipette, adding the mixed solution into DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) of the step 2.1, gently blowing and uniformly mixing by using a flaring gun head or lightly flicking a pipe wall by hand, uniformly mixing, and collecting the reaction solution to the pipe bottom by instantaneous centrifugation.

2.4 Split charging 360. Mu.L of the end repair reaction solution into 6 PCR tubes by using a flaring suction head, split charging 60. Mu.L of each tube, and collecting the reaction solution to the bottom of the tube by instantaneous centrifugation.

2.5 The PCR tube of 2.4 was placed in a PCR instrument and the reaction was performed according to the conditions of Table 2.

TABLE 2 reaction conditions for end repair plus "A

Temperature (temperature)	Time of
Thermal cover (105℃)	ON
20°C	10min
65°C	10min
4°C	Hold

2.6 Transient centrifugation the reaction was collected to the bottom of the tube and the 6 tube end-repaired plus "A" samples were pooled and transferred to a clean 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051).

3. End repair plus "A" product purification

3.1 Taking out Ampure XP magnetic beads (Beckman Coulter, A63882) from a 4-degree refrigerator 30min in advance, shaking and mixing uniformly, and then placing at room temperature, and shaking and mixing thoroughly before use.

3.2 Sucking 360 mu L of magnetic beads into a 2.6 sample, mixing the magnetic beads with a light bullet tube wall by hand, or blowing the magnetic beads with a flaring gun head for at least 6 times until the magnetic beads are completely mixed, and finally pumping all the liquid and the magnetic beads in the suction head into the tube.

3.3 Incubation on a rotary mixer for 5min at room temperature.

3.4 After the DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was centrifuged instantaneously, it was placed on a magnetic rack, left for 2-5 min until the liquid was clear, the supernatant was carefully aspirated with a pipette and discarded.

3.5 Holding DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) on a magnetic rack, adding 750. Mu.L of freshly prepared 80% ethanol to rinse the beads and tube walls, standing for 30s, carefully pipetting the supernatant and discarding.

3.6 Repeat step 3.5. And taking the centrifuge tube off the magnetic rack, performing instantaneous centrifugation, separating on the magnetic rack, and sucking the residual liquid at the bottom of the tube by using a small-range pipette.

3.7 Holding DNA LoBind Microcentrifuge Tube tubes (Eppendorf, 0030108051) on a magnetic rack, opening the centrifuge tube lid, and drying at room temperature until the surface of the beads is free of light reflection and cracking.

3.8 Remove DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) from the magnet holder, add 392. Mu.L of Nuclease-FREE WATER (Thermofisher, AM 9932) to elute DNA, mix well with the walls of a hand flick tube. The tube was centrifuged for 3 seconds and the liquid was collected at the bottom.

3.9 Incubation for 5min at room temperature.

3.10 After the DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was centrifuged instantaneously, it was placed on a magnetic rack, left for 2 to 5min until the liquid was clear, and 390. Mu.L of the supernatant was transferred to a new 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051). The remaining samples can be used for concentration determination, preferably using Qubit-DSDNA HS ASSAY KIT (Thermofisher, Q32854) for concentration determination of the purified end repair plus "A" product.

4. Joint connection

4.1 The metal bath temperature was set to 25 ℃ and preheating was performed.

4.2 The adaptor (Ad 3) and T4DNA ligase (NEB, E6057 AVIAL) were removed from the-20℃refrigerator, and the flick tube wall was mixed and centrifuged briefly and placed on ice. Quick Ligation Reaction Buffer (NEB, E6058 AVIAL) was thawed, air-blown, mixed, and then centrifuged briefly before being placed on ice. The ligation mixture was prepared according to Table 3.

TABLE 3 ligation reaction mixture

Component (A)	Single reaction volume
Rapid ligation reaction buffer (NEB, E6058 AVIAL)	120μL
T4DNA ligase (NEB, E6057 AVIAL)	60μL
Total volume of	180μL

4.3 To 3.10 a 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) containing 390. Mu.L of purified end repair plus "A" product was added 30uL of the Ad3 linker for vegetation in example 1. The mixture is gently beaten and evenly mixed for 6 times by a flaring suction head, and the reaction liquid is collected at the bottom of the tube by instantaneous centrifugation.

4.4A pipette is used to slowly aspirate 180. Mu.L of the prepared adaptor-ligation reaction mixture, the mixture is added into a 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) of step 4.3, gently swirled and mixed 6 times by a flaring tip, and the reaction mixture is collected at the bottom of the tube by instantaneous centrifugation.

4.5 The 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) from step 4.4 was placed in a 25℃preheated metal bath in 4.1 for the ligation reaction and a timer was run for 30min.

4.6, After the reaction, the reaction tube is instantaneously centrifuged, and the reaction solution is collected to the bottom of the tube.

5. Ligation product purification

5.1 Taking out Ampure XP magnetic beads (Beckman Coulter, A63882) from a 4 ℃ refrigerator 30min in advance, shaking and mixing uniformly, and then placing the mixture at room temperature, and fully shaking and mixing uniformly before use.

5.2 Sucking 240. Mu.L of magnetic beads into the 4.6 sample, mixing the mixture with the wall of a tube by using a light bullet, or blowing the mixture with a flaring gun head for at least 6 times until the mixture is completely mixed, and finally, ensuring that all liquid and magnetic beads in the suction head are driven into the tube.

5.3 Incubation on a rotary mixer for 5min at room temperature.

5.4 After the DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was centrifuged instantaneously, it was placed on a magnetic rack, left for 2-5 min until the liquid was clear, the supernatant was carefully aspirated with a pipette and discarded.

5.5 Holding DNA LoBind Microcentrifuge Tube tubes (Eppendorf, 0030108051) were placed on a magnetic rack, 900. Mu.L of Wash buffer1 (for short Fragment) or Wash buffer2 (for long Fragment) were added, DNA LoBind Microcentrifuge Tube tubes (Eppendorf, 0030108051) were removed from the magnetic rack, and the walls of the flick tubes were used to mix the beads. After mixing, the mixture is placed back to the magnetic rack, and kept stand for 2 to 5 minutes until the magnetic beads are all close to the wall, and the supernatant is carefully sucked and discarded.

5.6 Repeat step 5.5. And taking the centrifuge tube off the magnetic rack, performing instantaneous centrifugation, separating on the magnetic rack, and sucking the residual liquid at the bottom of the tube by using a small-range pipette.

5.7 Remove DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) from the magnetic rack, add 68. Mu.L Elution buffer (Elutation buffer) to elute DNA, mix by flick the tube wall with hand. The tube was centrifuged for 3 seconds and the liquid was collected at the bottom.

5.8 Incubation for 10min at room temperature, and 10min at 37℃when library inserts were longer.

5.9 After the DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was centrifuged instantaneously, it was placed on a magnetic rack, left for 2 to 5min until the liquid was clear, and 66. Mu.L of the supernatant was transferred to a new 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051). The remaining samples can be used for concentration determination, and it is recommended to use Qubit-DSDNA HS ASSAY KIT (Thermofisher, Q32854) for concentration determination of the purified ligation product.

6. Enzymatic digestion

6.1 The metal bath temperature was set to 37 ℃ and preheating was performed.

6.2T 7Exonuclease (T7 exonuclease; NEB, M0263 LVIAL) and NE Buffer ^TM 4 (NEB, B7004 SVIAL) were removed from the kit, vortexed 3 times for 3s each, and placed on ice after transient centrifugation. The enzyme digestion reaction mixture was prepared according to Table 4.

TABLE 4 enzyme digestion reaction mixture

Component (A)	Single reaction volume
NE Buffer ^TM 4(NEB,B7004SVIAL)	7.5μL
T7 Exonuclease(NEB,M0263LVIAL)	1.5μL
Total volume of	9μL

6.3 To 1.5mL DNA LoBind Microcentrifuge Tube containing 66. Mu.L of the purified ligation product in 5.9, 9. Mu.L of the enzyme digestion reaction mixture was added, gently swirled and mixed 6 times with a flaring tip, and the reaction mixture was collected at the bottom of the tube by instantaneous centrifugation.

6.4 The 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) from step 6.3 was placed in a 37℃preheated metal bath in 6.1 for ligation, and a timer was run for 5min.

6.5, After the reaction, the reaction tube is instantaneously centrifuged, and the reaction solution is collected to the bottom of the tube.

7. Purification of enzymatic digestion products

7.1 Taking out Ampure XP magnetic beads (Beckman Coulter, A63882) from a 4 ℃ refrigerator 30min in advance, shaking and mixing uniformly, and then placing the mixture at room temperature, and fully shaking and mixing uniformly before use.

7.2 Sucking 75. Mu.L of magnetic beads into a 6.5 sample, mixing the mixture with a light bullet tube wall by hand, or blowing the mixture with a flaring gun head for at least 6 times until the mixture is completely mixed, and finally, ensuring that all liquid and magnetic beads in the suction head are driven into the tube.

7.3 Incubation on a rotary mixer for 5min at room temperature.

7.4 After the DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was centrifuged instantaneously, it was placed on a magnetic rack, left for 2-5 min until the liquid was clear, the supernatant was carefully aspirated with a pipette and discarded.

7.5 Holding DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) placed on a magnetic rack, 200. Mu.L Wash buffer1 (for short Fragment) was added, DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was removed from the magnetic rack and the walls of the flick tube were homogenized. After mixing, the mixture is placed back to the magnetic rack, and kept stand for 2 to 5 minutes until the magnetic beads are all close to the wall, and the supernatant is carefully sucked and discarded.

7.6 Repeat step 7.5. When a small amount of liquid remains on the tube wall, the centrifugal tube can be instantaneously centrifuged, and after the liquid is separated on the magnetic rack, the liquid at the bottom of the tube is sucked by a small-range pipette.

7.7 Remove DNA LoBind Microcentrifuge Tube tubes (Eppendorf, 0030108051) from the magnet holder, add 62. Mu.L of Elution buffer (Elution buffer) to elute DNA, mix well with the walls of a hand flick tube. The tube was centrifuged for 3 seconds and the liquid was collected at the bottom.

7.8 Incubation for 10min at room temperature, 37℃incubation was used when the library was too long.

7.9 After the DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) was centrifuged instantaneously, it was placed on a magnetic rack, left for 2 to 5min until the liquid was clear, and 60. Mu.L of the supernatant was transferred to a new 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051). The remaining samples can be used for concentration determination, and it is recommended to use Qubit-DSDNA HS ASSAY KIT (Thermofisher, Q32854) for concentration determination of the purified enzyme digestion product.

7.10 To complete the whole warehouse-building process, the product can be stored in a 4-degree refrigerator for 72 hours.

7.11 The above library construction procedure was performed using a plasmid restriction enzyme cleavage product as a quality control, and the comparison results before and after the restriction enzyme cleavage showed that three forms of molecules exist after ligation, which were original templates to which no upper linker was attached, and a library to which single-ended linkers and both-ended linkers were attached, respectively. After exonuclease treatment, the non-ligated and single-ended-ligated products were significantly reduced, and as the cleavage time increased, the ligated double-ended-ligated products did not significantly decrease, indicating that this step of enzyme treatment was effective in enriching the target double-ended-ligated products (FIG. 6).

Example 4 construction of a sequencing library without an exonuclease step

The complete flow of the embodiment of the invention is shown in fig. 2.

DNA interruption (optional)

If the initial genome input is less than 12. Mu.g, DNA may be fragmented to increase the library capture rate during on-press sequencing. It is recommended to use Covaris g-TUBE (Covaris, 520079) for fragmentation breaks, breaking the system and steps reference g-TUBE instructions.

2. End repair plus "A"

89122.1 In 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051), the total DNA was calculated from the sample concentration to be 12 μg of the volume of sample to be added, and the complementary nucleic-FREE WATER (Thermofisher, AM 9932) homogenizes the sample volume to 288 μl.

2.2 According to the ratios in Table 5, the end repair plus "A" reaction mixture was prepared in a centrifuge tube in the amount required for detection. The preparation is carried out on ice, the prepared end repair is added with the 'A' reaction mixed solution to vortex and shake for 3 times, 3s each time, and the reaction solution is collected to the bottom of the tube by instantaneous centrifugation.

TABLE 5 reaction mixture of end repair and "A

2.5 The PCR tube of 2.4 was placed in a PCR instrument and the reaction was performed under the conditions of Table 6.

TABLE 6 reaction conditions for end repair plus "A

3. End repair plus "A" product purification

3.3 Incubation on a rotary mixer for 5min at room temperature.

3.6 Repeat step 3.5. And taking the centrifuge tube off the magnetic rack, performing instantaneous centrifugation, separating on the magnetic rack, and sucking residual liquid at the bottom of the centrifuge tube by using a small-range pipette.

3.9 Incubation for 5min at room temperature.

4. Joint connection

4.1 The metal bath was set to 25 ℃ and preheated.

4.2 The adaptor (Ad 3) and T4DNA ligase (NEB, E6057 AVIAL) were removed from the-20℃refrigerator, and the flick tube wall was mixed and centrifuged briefly and placed on ice. Quick Ligation Reaction Buffer (NEB, E6058 AVIAL) was thawed, air-blown, mixed, and then centrifuged briefly before being placed on ice. The ligation mixture was prepared according to Table 7.

TABLE 7 ligation reaction mixture

4.3 To 3.10 a 1.5mL DNA LoBind Microcentrifuge Tube tube (Eppendorf, 0030108051) containing 390. Mu.L of purified end repair plus "A" product was added 30uL of the Ad3 linker prepared in example 1. The mixture is gently beaten and evenly mixed for 6 times by a flaring suction head, and the reaction liquid is collected at the bottom of the tube by instantaneous centrifugation.

5. Ligation product purification

5.1 Taking out Ampure XP magnetic beads (Beckman Coulter, A63882) from a 4-degree refrigerator 30min in advance, shaking and mixing uniformly, and then placing at room temperature, and shaking and mixing thoroughly before use.

5.3 Incubation on a rotary mixer for 5min at room temperature.

Example 5 sequencing library preparation

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 libraries that were finally used in a sequencing machine.

1. To 100mL of 1M Tris-HCl pH 7.5 buffer and 100mL of 1M KCl solution in a volumetric flask were added ultrapure water to a constant volume of 1L to prepare a 2X binding buffer.

2. A mixed solution of helicase Dda (prepared from example 2) and library was prepared on ice according to Table 8 below, followed by incubation at 30℃for one hour.

TABLE 8 Motor protein and library binding System

Reagent(s)	Volume of
2X binding buffer	50μL
Helicase Dda	20μL
Ligation products after purification	20μL
Water and its preparation method	10μL

3. The library was quantitated using the Qubit DNA HS kit and after labeling for clear concentration, the product was stored in a4 ℃ refrigerator for use.

EXAMPLE 6 nanopore sequencing

In this embodiment, nanopore sequencing is performed on the target sequencing library prepared in embodiment 6 based on the nanopore detection platform of the patch clamp platform, so as to verify the advantage of the constructed sequencing library in nanopore sequencing, namely the increase of the number of molecules to be sequenced.

1. Referring to Geng Jia, guo Peixuan ("application of phage phi29DNA packaging motor phospholipid membrane chimera in single molecule detection and nano medical field". Life sciences 2011,23 (11): 1114-1129), a nanopore detection platform based on a patch clamp platform is built, and porin (Sigma-Aldrich, H9395-5 mg) is inserted onto a phospholipid bilayer membrane to form a single channel nanopore.

2. The sequencing library obtained in example 5 was added to the single channel system and the current amplitude changes were detected and recorded by the patch clamp system.

3. As shown in FIG. 7, the current changes in the unit time of library sequencing in example 3 and example 4 show that the library sequencing sequence after enzyme digestion treatment is obviously increased in the unit time, the free linker sequence signal is reduced, the occupied time of resting potential is reduced, and the sequencing flux is obviously improved.

4. Comparison of the free linker ratios of the original library and the digested library shows that after digestion, the measured free linker ratio is reduced from 41.46% to 8.82% of the original library, and the library molecular ratio is increased from 58.54% to 91.18% (FIG. 8).

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

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

A method of creating a sequencing library, comprising:

Digestion of the adaptor-ligated test sample with exonuclease to obtain the sequencing library.
The method of claim 1, further comprising subjecting the digested product to a de-spinning process;

optionally, the unwinding treatment is performed under the action of a helicase.
The method according to claim 2, wherein the helicase is 5 'to 3' in the direction of DNA movement and the exonuclease is a 5'- >3' exonuclease;

Optionally, the 5'- >3' exonuclease recognizes double-stranded DNA, the exonuclease comprising at least one selected from T7 exonuclease, lambda exonuclease, T5 Exonuclease, exonuclease VI, exonuclease VIII (truncated);

Preferably, the exonuclease is T7 exonuclease.
The method according to claim 2, wherein the helicase is 3 'to 5' in the direction of DNA movement and the exonuclease is a 3'- >5' exonuclease;

Optionally, the 3'- >5' exonuclease recognizes double-stranded DNA, the exonuclease comprising at least one selected from Exonuclease III, exonuclease IX, exonuclease X.
The method of claim 1, wherein the exonuclease recognizes single-stranded DNA.
The method of claim 1, further comprising performing degradation-resistant modification on the 5 'end and/or the 3' end of the sample to be tested to which the linker is attached;

Optionally, the degradation preventing modification comprises at least one selected from the group consisting of a phosphate modification, a 2'-OH modification (RNA base), a 2' -F modification, an LNA lock nucleotide modification, and a PNA peptide nucleic acid modification.
The method of claim 1, wherein the digestion is carried out at 37 ℃ for 4-6 minutes at a ligation product to T7 exonuclease ratio of 44:1.
The method of claim 1, wherein the linker is a Y-linker or a non-Y-linker;

Optionally, the non-Y-type linker has at least one of the following structures:

complete complementary duplex, complementary duplex-non-complementary single strand-complementary duplex, 5 'overhanging single strand-complementary duplex, 3' overhanging single strand-complementary duplex.
The method of claim 1, further comprising purifying the digested product, optionally, using Ampure XP magnetic beads.
The method according to claim 1, wherein the sample to be tested to which the adaptor is attached is obtained by:

Performing terminal repair and A adding treatment on a sample to be tested;

carrying out joint connection treatment on the A-added treatment product so as to obtain the sample to be tested, wherein the sample is connected with the joint;

optionally, after the end repair and the addition a treatment and before the ligation treatment, further comprising subjecting the addition a treated product to a first purification treatment;

Optionally, after the ligation treatment, further comprising subjecting the ligation treatment product to a second purification treatment.
The method of claim 1, wherein the sample to be tested is a DNA sample.
The method of claim 1, further comprising fragmenting the test sample prior to subjecting the test sample to the end repair and a-addition process.
A sequencing library, characterized in that it is obtained by the method of any one of claims 1 to 12.
A sequencing method, which is characterized by comprising the step of sequencing the sequencing library according to claim 13 so as to obtain the sequence of a sample to be tested;

optionally, the sequencing is performed on a nanopore sequencing platform.
A kit for constructing a sequencing library is characterized by comprising a reagent, wherein the reagent comprises exonuclease and is used for digestion treatment of a sample to be tested connected with a connector;

Optionally, further comprising a first reagent adapted to fragment the sample to be tested;

Optionally, further comprising a second reagent adapted to perform end repair and a-addition treatment on the sample to be tested;

Optionally, a third reagent is further included, the third reagent being adapted to subject the sample to be tested to a linker ligation process.
The kit of claim 15, further comprising a linker carrying a 5' terminal phosphate group.
The kit of claim 16, wherein the exonuclease comprises at least one of T7 exonuclease,Lambda exonuclease,T5 Exonuclease,Exonuclease VI,Exonuclease VIII(truncated),Exonuclease III,Exonuclease IX,Exonuclease X.