WO2018090373A1 - Method for repairing dna terminal end and adding a - Google Patents

Abstract

Provided is a method for repairing a DNA terminal end and adding A, said method comprising: in the same reaction system, in the presence of dNTP, using a polymerase to blunt or flatten a fragmented DNA terminal end, and using a polynucleotide kinase to transform a 5'-hydroxyl into a 5'-phosphate group and transform a 3'-phosphate group into a 3'-hydroxyl; in the presence of excess dATP, using a polymerase not having exonucleolytic activity to add dATP to a double-stranded DNA 3' terminal end. Using the method, a DNA terminal end is repaired and A is added in the same reaction system, thus simplifying enzyme reaction and purification steps, increasing the efficiency of transforming starting DNA into ligatable adapter DNA, and reducing the requirement of the total amount of starting DNA.

Description

Method for DNA end repair and addition of A Technical field

The invention relates to the technical field of molecular biology, in particular to the field of library construction technology, in particular to a method for DNA end repair and addition of A and a method for DNA interruption, end repair and addition of A, and library construction method based thereon .

Background technique

With the rapid development of next-generation sequencing technology, high-throughput sequencing technology is widely used in biology, medicine and other industries, promoting the development of biomolecular mechanisms and biotechnology. Library construction methods based on high-throughput sequencing technology are also being continuously improved and optimized to meet the requirements of different sample sizes, different species and different sequencing purposes. In general, the core steps of library construction of high-throughput sequencing technology include: fragmenting genomic DNA, enzymatically repairing DNA damage, ligating the ligation link at the double end of the DNA, and amplifying the target DNA ligated with the linker for sequencing. Library.

Existing library construction methods involve multiple steps, in particular DNA disruption, end repair and addition of A in a number of different steps, so the steps are cumbersome, less efficient and require a higher initial sample size. It is imperative to simplify the library construction process and improve the success rate of database construction with low initial samples. By optimizing and improving the library construction method, shortening the enzyme reaction time and building the library steps, and improving the efficiency of the sample conversion into the library become a key point in the application field of high-throughput sequencing technology.

In addition, existing library construction methods, specifically to the DNA disruption step, also have some problems. Common methods of DNA disruption are physical disruption and enzyme disruption. The physical interruption method is exemplified by the commonly used Covaris interrupter. Covaris interrupters work by breaking the DNA with sound waves. The whole process involves placing the DNA in a low-temperature bath environment, and obtaining DNA fragments of different sizes by adjusting the intensity, duration, and surface medium that changes the DNA contact. The disadvantages of this method are as follows: First, the pre-cooling interrupter takes a long time, usually takes at least 30 minutes; secondly, it is not conducive to the development of a fully automated database; third, the DNA starting amount is high (at least) 100ng), is not conducive to the development of micro-building. The enzyme breaking method is exemplified by NEB's interrupting kit. The kit uses a mixture of two DNA endonucleases to create a gap in the double-stranded DNA by a non-limiting DNA endonuclease, and another DNA endonuclease cleaves the corresponding strand of the gap to effect fragmentation. Although the enzyme disruption method can effectively fragment genomic DNA, the fragment concentration is interrupted at a low initial amount, and the non-limiting DNA endonuclease has a base preference, which affects the uniformity of sequencing data coverage. .

Summary of the invention

The invention provides a method for DNA end repair and addition of A, which can realize DNA end-repair and A-reaction in the same reaction system, simplify enzyme reaction and purification steps, and improve conversion of starting DNA into connectable linker DNA. Efficiency, reducing the requirement for the total amount of starting DNA. Further, this issue Ming provides a DNA interrupt-end repair-plus A method, which can achieve DNA disruption, end repair and A reaction in the same reaction system, further simplifying the enzyme reaction and purification steps. The invention also provides a reaction system and a kit for DNA end repair and addition of A.

According to a first aspect of the present invention, the present invention provides a method for DNA end repair and addition of A, which comprises: filling or cutting the ends of the fragmented DNA by using a polymerase in the same reaction system in the presence of dNTP Ping, using a polynucleotide kinase to convert a 5' hydroxyl group into a 5' phosphate group and a 3' phosphate group into a 3' hydroxyl group; in the presence of excess dATP, there is no 3'-5' exoactivity The polymerase adds dATP to the 3' end of the double stranded DNA.

According to a second aspect of the present invention, the present invention provides a library construction method based on DNA end repair and addition of A, the method comprising the steps of: using a polymerase to fragment DNA in the same reaction system in the presence of dNTP The ends are filled or squashed, and the 5' hydroxyl group is converted into a 5' phosphate group by a polynucleotide kinase and the 3' phosphate group is converted into a 3' hydroxyl group; in the presence of excess dATP, the use does not have 3'- The 5' exo-active polymerase adds dATP to the 3' end of the double-stranded DNA; in the above reaction system, the linker and the ligation reaction mixture are directly added to make the 3'A protruding double-stranded DNA produced in the previous step and the above-mentioned linker connection.

According to a third aspect of the present invention, there is provided a method of DNA disruption-end repair-addition A comprising: using a non-limiting DNA endonuclease on DNA in the same reaction system in the presence of dNTP A nick or gap is formed while nick translation or strand displacement is performed using DNA polymerase and end-repairing; dATP is added to the 3' end of the double-stranded DNA using DNA polymerase in the presence of excess dATP.

According to a fourth aspect of the present invention, the present invention provides a DNA disruption-end repair-addition A method, which comprises: in the same reaction system, using a disruption enzyme to randomly break a DNA fragment in the presence of dNTP, while simultaneously Terminal repair using DNA polymerase; end-addition of dATP reaction using DNA polymerase in the presence of excess dATP.

According to a fifth aspect of the present invention, the present invention provides a library construction method based on DNA break-end repair-plus A, which comprises the steps of: using non-limiting DNA in the same reaction system in the presence of dNTP Endonuclease forms a nick or gap in DNA, and uses DNA polymerase for nick translation or strand displacement and end-repair; in the presence of excess dATP, DNA polymerase is used to add dATP to the 3' end of double-stranded DNA; In the reaction system, a linker and a reaction mixture are directly added, and the double-stranded DNA produced in the previous step is linked to the above-mentioned linker.

According to a sixth aspect of the present invention, the present invention provides a library construction method based on DNA break-end repair-plus A, which comprises the steps of: randomly using a disruption enzyme in the presence of dNTP in the same reaction system The DNA fragment is interrupted, and the DNA polymerase is used for terminal repair; in the presence of excess dATP, the DNA polymerase is used for the terminal dATP reaction; in the above reaction system, the linker and the reaction mixture are directly added to make the previous step. Double-stranded DNA is ligated to the above linker.

According to a seventh aspect of the present invention, the present invention provides a reaction system for DNA end repair and addition of A, The system is contained in every 50 μL of the system, physically interrupting DNA 1-100 ng, T4 DNA polymerase 1.2-10 U, Klenow large fragment 0-2 U, T4 polynucleotide kinase 4-16 U, Taq polymerase 1-4U , each dNTP each 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg ions 8-15 mM; or, the system is contained in every 50 μL of the system, plasma free DNA or enzyme digestion to interrupt DNA 1-100ng, T4 DNA polymerase 0-3U, Klenow large fragment 0-2U, T4 polynucleotide kinase 4-10U, Taq polymerase 1-2U, each dNTP 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg The ion is 8-10 mM, provided that the content of the T4 DNA polymerase and the large Klenow fragment are different.

According to an eighth aspect of the present invention, there is provided a kit for DNA end-repair and addition of A according to T4 DNA polymerase 1.2-10 U, Klenow large fragment 0-2 U, T4 polynucleotide kinase 4-16 U, Taq polymerase 1-4 U, each 1-10 nmol of each dNTP, additional dATP 20-50 nmol, and Mg ion 400-750 nmol, forming a kit unit for dilution into a 50 μL reaction system; or, the reagent The cassette is according to T4 DNA polymerase 0-3U, Klenow large fragment 0-2U, T4 polynucleotide kinase 4-10U, Taq polymerase 1-2U, each dNTP each 1-10nmol, additional dATP 20-50nmol, And Mg ions of 400-500 nmol, provided that the content of the T4 DNA polymerase and the large Klenow fragment are different, forming a kit unit for dilution into a 50 μL reaction system.

According to a ninth aspect of the present invention, the present invention provides a reaction system for DNA disruption-end repair-addition A, which is contained in every 30 μL of the system, 5-100 ng of genomic DNA, and 0.5-3 U of NEB interrupting enzyme. Polymerase 0-20U with 3'-5' exo-activity or 0-20U with chain-replacement activity, polymerase 1-15U without 3'-5' exo-activity, 0.02 for each dNTP -0.2 mM, additional dATP 0.4-1 mM, and Mg ion 8-15 mM; alternatively, the system is contained in every 30 μL of system, genomic DNA 5-100 ng, non-limiting DNA endonuclease 0.5-3 U, with 3'- 5' exo-active polymerase 0-20U or/and polymerase 0-20U with strand displacement activity, 1-15U without 3'-5' exo-activity, 0.02-0.2 mM for each dNTP, Additional dATP 0.4-1 mM, and Mg ions 8-15 mM.

According to a tenth aspect of the present invention, the present invention provides a DNA interrupt-end repair-plus A reaction kit, which is 5-3 ng in genomic DNA, and NEB interrupts 0.5-3 U, having 3'-5 'Exo-active polymerase 0-20U or/and polymerase 0-20U with strand displacement activity, 1-15U without 3'-5' exo-activity, 0.02-0.2 mM per dNTP, extra dATP 0.4-1 mM, and Mg ion 8-15 mM, forming a kit unit for dilution into a 30 μL reaction system; or, the kit is 5-100 ng according to genomic DNA, non-limiting DNA endonuclease 0.5-3 U, with 3 '-5' exo-active polymerase 0-20U or/and polymerase 0-20U with strand displacement activity, polymerase 1-15U without 3'-5' exo-activity, 0.02-0.2 per dNTP mM, additional dATP 0.4-1 mM, and Mg ions 8-15 mM, A kit unit for dilution into a 30 μL reaction system was formed.

It should be noted that the kit of the present invention does not mean that the kit of the present invention is limited to a kit for dilution into a 50 μL or 30 μL reaction system, but the kit of the present invention is determined according to the above definition. The proportional relationship and concentration of the various components. Those skilled in the art can further enlarge or reduce the capacity of the kit in parallel according to the proportional relationship and concentration of various components in the kit unit for dilution into 50 μL or 30 μL of the reaction system, as long as the ratio of each component is The relationship and concentration are in agreement with the above definitions and are considered to be kits of the present invention. The above kit units are artificially defined, and those skilled in the art will appreciate that the capacity of a kit that is enlarged or reduced in parallel may be referred to as a kit unit.

DRAWINGS

1 is a schematic diagram showing the process of constructing a terminal repair-plus A-plus-linkage reaction method based on the BGISEQ-500/1000 sequencing platform according to an embodiment of the present invention (ie, "one-tube method" in the figure); DNA; 2: end repair and addition of A in the same reaction system; 3.1: using PCR strategy, both ends of the DNA plus bubbling type connector (excluding U base); 3.2: using PCR-free (no PCR) In strategy, a buccal-type linker containing a U base is added to both ends of the DNA, and USER treatment is performed at the same time. The 5' end of the DNA of the USER-treated linker is a phosphate group to facilitate subsequent single-chain cyclization; 4: Purification reaction System, remove joint contamination; 5: For PCR construction strategy, connect products for PCR amplification; for PCR-free database construction strategy, the ligation product is used directly for single-chain cyclization; 6: Purify PCR reaction system to remove primer dimerization Body contamination; 7: PCR products were used for single-chain cyclization.

2 is a schematic diagram of a database construction process of an end-repair-addition A-plus-linkage reaction method based on an Illumina sequencing platform according to another embodiment of the present invention; wherein: 1: starting DNA; 2: terminal repairing and adding A in the same reaction system In the PCR strategy, the Y-type linker containing the tag sequence is added to both ends of the DNA; 3.2: When the PCR-free strategy is used, the Y-type linker containing the tag sequence is added to both ends of the DNA; 4: Purification reaction The system removes the contamination of the linker; for the PCR-free database construction strategy, the purified ligation product can be directly used in the machine after quantification by Q-PCR; 5: PCR amplification of the ligation product for PCR construction strategy; 6: Purification PCR The reaction system removes primer dimer contamination, and the purified PCR product can be directly used in the machine after quantification by Q-PCR.

3 is a schematic diagram of a library construction process of a break-end repair-plus A one-step method based on CG (Complete Genomics) sequencing platform according to another embodiment of the present invention; wherein: 1: unrestricted endonuclease or disruption enzyme Cooperates with DNA polymerase to fragment (ie, interrupt) DNA and complete end-repair, and add adenyl deoxyribonucleic acid (dATP) at the 3' end; 2: DNA fragment to the bubble linker; 3: PCR strategy, PCR amplification increases the DNA fragment of the buccal linker; 4: For the PCR-free strategy, USER digests the DNA fragment; 5: cyclizes the single-stranded DNA to generate the final product of the constructed library.

4 is a break-end repair-addition based on an Illumina sequencing platform according to another embodiment of the present invention. A one-step library construction flow diagram; wherein, 1: unrestricted endonuclease or disruption enzyme cooperates with DNA polymerase to fragment (ie, interrupt) DNA and complete end repair, and at the 3' end Plus adenyl deoxyribonucleic acid (dATP); 2: DNA fragment linked to Y linker; 3: PCR amplification increases the product of the linker.

5 shows an electrophoresis pattern of PCR products of 8 human plasma samples according to an embodiment of the present invention. The fragment size of the PCR product is about 250 bp, which meets the requirements of the BGISEQ-500/1000 sequencing platform.

FIG. 6 shows the results of PCR-Free library DNA nanosphere electrophoresis according to an embodiment of the present invention. Most of the samples are in the pores and cannot be electrophoresed, and the DNA nanospheres cannot be run out of the gel during polyacrylamide gel electrophoresis. The characteristics of the hole.

7 shows a sequencing depth distribution map of a genomic sample according to an embodiment of the present invention. The sequencing depth is in accordance with the Poisson distribution, and the sequencing depth is concentrated at about 30×, which is in line with the data requirements of human genome resequencing.

Figure 8 is a graph showing the cumulative ratio of GC content of a genomic sample according to one embodiment of the present invention. Although the coverage of fragments with high GC content is decreased, the coverage is close to 1, indicating that most of the fragments with high GC content can be It was detected that the coverage of all fragments with different GC contents was substantially maintained.

9 shows an electrophoresis pattern of a PCR product of 8 enzyme-interrupted genomic samples according to an embodiment of the present invention. The fragment size of the PCR product is about 250 bp, which meets the requirements of the BGISEQ-500/1000 sequencing platform.

Figure 10 shows the results of bio-analysis 2100 of PCR products of two enzyme-interrupted genomic samples according to one embodiment of the present invention, the fragment size of the PCR product being about 250 bp.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

In a technical solution of the present invention, a method for DNA end repair and addition of A is proposed, which can realize DNA end repair and addition of A in the same reaction system (one tube), and can be directly in the reaction tube. The joint is connected and the reaction mixture is connected to realize a one-tube type joint.

According to one aspect of the present invention, the method for repairing and adding A to the DNA includes: in the same reaction system, using a polymerase to fill or squash the ends of the fragmented DNA in the same reaction system, using a polynucleoside Acid kinase converts a 5' hydroxyl group to a 5' phosphate group and a 3' phosphate group to a 3' hydroxyl group; in the presence of excess dATP, a double stranded DNA 3 is used in a polymerase that does not have a 3'-5' exo-activity 'end plus dATP.

The DNA end-repairing and A-added DNA products obtained by the method of DNA end repair and addition of A of the present invention can be applied to various application scenarios, and the most common application is for library construction. By adopting the DNA end-repairing and A-reaction method of the invention, it is possible to achieve rapid establishment of a low initial amount of interrupted samples or plasma free DNA, shorten the time of library preparation, reduce the cost of library preparation, and increase the available number of libraries. According to the output, and not only can be applied to the BGISEQ platform, it can also be extended to Illumina or other sequencing platforms that use 3'T protruding connectors as a library connector. In the present invention, the 3'T protruding joint may be a bubbling type joint, a Y type joint, a neck ring type joint or any other 3'T protruding joint.

In another technical solution of the present invention, a DNA interruption-end repair-addition A method is proposed, which can realize DNA interruption, end repair and addition of A in the same reaction system (one tube). Moreover, the joint and the reaction mixture can be directly added to the reaction tube to realize a one-tube type joint.

The technical solutions of the present invention will be described in detail below by taking different platforms as an example and referring to the accompanying drawings. It should be noted that the platforms referred to below are merely exemplary, and the present invention is not limited to these platforms, but can be applied to a wider range of platforms. And the technical solution of the present invention is not limited to the details of the technical solutions described below with reference to the drawings.

Referring to FIG. 1, according to the first embodiment of the present invention, the library construction of the BGISEQ-500/1000 sequencing platform is taken as an example, and the specific steps of the library construction process of the present scheme are as follows:

1. Preparation of template DNA: The library DNA is prepared by using the prepared template DNA, including but not limited to plasma free DNA, ChIP DNA, FFPE DNA, ultrasonic disruption or enzyme digestion to break the purified genomic DNA or RNA. The cDNA obtained by transcription (shown as number 1 in Figure 1).

2. DNA end-repair and A-reaction: in the same reaction system, in the presence of dNTP, the ends of the fragmented DNA are filled or squashed by polymerase, for example, using the 5'-3' polymerization activity of the polymerase. 'The overhanging end-filling and/or 3'-5' exo-activity of the polymerase cleaves the 3' overhang, using the polynucleotide kinase to convert the 5' hydroxyl group to a 5' phosphate group and the 3' phosphate group The group is converted to a 3' hydroxyl group; in the presence of excess dATP, dATP is added to the 3' end of the double-stranded DNA using a polymerase having no 3'-5' exo-activity (as indicated by number 2 in Figure 1).

The integrity of the ends of the genomic DNA fragments generated by enzymatic cleavage by the body itself or by humans physically or chemically disrupted by the physical or chemical methods will be disrupted to varying degrees, forming 5' overhangs, 3' overhangs, 5' hydroxyls, The configuration of the 3' phosphate group is not conducive to subsequent attachment reaction with the linker. The end-repair reaction mainly utilizes the 5'-3' polymerization activity of a polymerase (such as T4 DNA polymerase, Klenow large fragment, etc.) in the presence of deNTP-ribonucleoside triphosphate (deoxy-ribonucleoside triphosphate) to fill the 5' overhang end. , 3'-5' exonuclease activity cleaves the 3' overhang; the 5' hydroxy kinase activity of a polynucleotide kinase (eg, T4 polynucleotide kinase, etc.) converts the 5' hydroxyl to 5' The phosphate group, the 3' phosphatase activity of the polynucleotide kinase converts the 3' phosphate group to a 3' hydroxyl group. The 3' end plus A reaction is the use of a polymerase that does not have 3'-5' exo-activity when a dATP is present in the reaction system (eg, a large Klenow fragment with 3'-5' exo-activity removed (also known as " Klenow fragment (3'→5'exo-)"), Taq DNA polymerase without 3'-5' corrective activity, etc.), add dATP at the 3' end of double-stranded DNA to facilitate double-stranded DNA follow-up with 3' The linker sequence of the T overhang configuration is AT linked.

Based on the above principle, the end-repair and A-addition reactions are carried out in the same reaction system, such as polymerase, polynucleotide kinase, polymerase without 3'-5' exo-activity, and dNTP (in which dATP is required) In dTTP, dCTP, dGTP), a reaction buffer compatible with three enzymes.

In a preferred embodiment of the present invention, the enzyme amount, the reaction buffer component, the reaction temperature, and the reaction time are systematically optimized, and a series of end portions suitable for different starting amounts and different source template DNAs are obtained. Repair the reagent formulation and storage conditions with A. Thus, by adding an optimized enzyme reaction mixture, the double-stranded DNA end-repair reaction and the 3'-end plus A reaction are sequentially carried out in one reaction system by controlling the reaction temperature.

Specifically, for physically interrupting DNA, every 100 μL of the system contains, physically interrupting DNA 1-100 ng, T4 DNA polymerase 1.2-10 U, Klenow large fragment 0-2 U, T4 polynucleotide kinase 4-16U Taq polymerase 1-4U, each dNTP 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg ion 8-15 mM; preferably, the reaction conditions are 15-37 ° C reaction 10-30 min, then 65-75 The reaction at °C for 10-30 min.

For plasma free DNA or digested DNA, every 50 μL of the system contains, plasma free DNA or digested DNA 1-100 ng, T4 DNA polymerase 0-3U, Klenow large fragment 0-2 U, T4 polynucleoside Acid kinase 4-10U, Taq polymerase 1-2U, each dNTP 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg ion 8-10 mM, provided that the content of T4 DNA polymerase and Klenow large fragment is different 0; preferably, the reaction conditions are 15-37 ° C for 10-30 min, then 65-75 ° C for 10-30 min.

It should be noted that the additional dATP 0.4-1 mM means that in addition to 0.02-0.2 mM each of dNTPs (including dATP, dTTP, dCTP, and dGTP), excess dATP 0.4-1 mM is contained. That is, the total amount of dATP is 0.42-1.2 mM.

It should be noted that the present invention is described by taking a 50 μL system as an example, and does not indicate that the method of the present invention is only suitable for a 50 μL system, and those skilled in the art can understand that the system is maintained based on the 50 μL system of the present invention. The present invention can be practiced by carrying out the parallel enlargement or reduction of the system, and the technical solution of the present invention also includes the parallel enlarged or reduced system described herein.

In another more preferred embodiment of the present invention, systematic optimization is carried out in terms of enzyme amount, reaction buffer component, reaction temperature, and reaction time, and a series of template DNAs suitable for different starting amounts and different sources are obtained. The end of the repair and addition of A reagent formulation and storage conditions (as shown in Table 1).

Table 1 End-repair of different template DNA in 50 μL reaction system-optimization parameters of reaction with A

It should be noted that in Table 1, for the enzyme digestion and plasma free DNA, the content of T4 DNA polymerase and Klenow large fragment is 0.

In still another more preferred embodiment of the present invention, systematic optimization is carried out in terms of enzyme amount, reaction buffer component, reaction temperature, and reaction time, and a series of DNA interrupts suitable for different starting amounts are obtained. End-repair, A reagent formulation and storage conditions (as shown in Table 2).

Table 2 Optimization parameters of the interruption, end repair and reaction of different template DNA under 30μL reaction system

It should be noted that in Table 2, the polymerase having the 3'-5' exo-activity is 0 when the content of the polymerase having the strand displacement activity is different; when the NEB-interrupting enzyme and the non-limiting DNA endonuclease are different, 0; a polymerase having a 3'-5' exo-activity such as T4 DNA polymerase or DNA polymerase I; a polymerase having strand displacement activity such as a large Klenow fragment; a polymerase having no 3'-5' exo-activity For example, the rTaq enzyme.

3. Add a linker: directly add the linker and the reaction mixture to the reaction solution of the step 2, and connect the double-stranded DNA protruding from the 3'A of the previous step to the linker. For different sequencing platforms, connectors for different platforms can be added.

A linker is a specially designed DNA sequence that is ligated to both ends of a DNA fragment by ligation, etc., and can be identified during sequencing and used as a starting site for sequencing, for the instrument to read subsequent sequence information. Due to the different library structures of different platforms, the joint structure used is also different. Using different adapters at this step will meet the needs of libraries for different sequencing platforms.

For the BGISEQ platform, when using the PCR database construction strategy, add a U-free bubbling type connector (as shown in Figure 3.1 in Figure 1); when using the PCR-free database construction strategy, add a U-containing bubbling type connector. At the same time, USER enzyme was added to carry out the digestion reaction, and the 5' end of the DNA which is ligated to the linker was digested by USER to produce a 5' phosphate group (as shown by the number 3.2 in Fig. 1).

4. Purification of the linker product: purification of the end-repair-plus A-plus linker reaction system to remove joint contamination (shown as number 4 in Figure 1).

5. PCR amplification or single-strand cyclization: For the PCR construction strategy, a nucleic acid single strand complementary to both ends of the target linker sequence is added as a primer for PCR amplification, and a large amount of DNA product is obtained, since one of the primers has a 5' end Phosphorylation, amplification of the double-stranded DNA has a phosphate group at the 5' end; however, for the PCR-free database, the purified ligation product is used directly for single-strand cyclization, the two strands of DNA Both can form a cyclized product (as indicated by number 5 in Figure 1).

6. Purification of the PCR reaction system: For the PCR construction strategy, the PCR reaction system is purified to remove the primer dimer contamination (shown as number 6 in Figure 1).

7. Single-chain cyclization: For the PCR database construction strategy, one of the chains of the purified PCR amplification product DNA can undergo a single-chain cyclization reaction (as shown by number 7 in Figure 1).

Single-strand cyclization uses thermal denaturation to convert double-stranded DNA into single-stranded deoxynucleic acid, and a single-stranded nucleic acid complementary to the head-to-tail sequence (which can be referred to as a mediated bridge sequence) is hybridized with the degenerated single-stranded deoxynucleic acid and The ligation reaction cyclizes the single-stranded nucleic acid of interest. The reacted single-chain cyclization system can be directly used for subsequent rolling circle replication to form a sequencing template product DNB (DNA Nanoball, nucleic acid nanosphere) for sequencing reaction.

Referring to FIG. 2, according to a second embodiment of the present invention, the library construction of the Illumina sequencing platform is taken as an example to illustrate the technical solution thereof.

As shown in FIG. 2, it differs from the library construction method of the BGISEQ-500/1000 sequencing platform shown in FIG. 1 in that, in the process of adding a linker in step 3, when a PCR database construction strategy is adopted, a label-free sequence is added. Y-type connector (as shown in Figure 3.1 in Figure 2); when using the PCR-free database construction strategy, add Enter the Y-junction with the tag sequence (as shown in Figure 3.2 in Figure 2). In addition, in the method shown in Figure 2, the final single-chain cyclization is not required, and the purified product can be directly used for sequencing on the machine.

The method for building the library of the present invention simplifies the steps 3 to 4 of the classical database construction method from the original 3-step enzymatic reaction and the 3-step purification reaction to a 2-step enzymatic reaction and a 1-step purification reaction, thereby greatly shortening the time for building the library and saving the purification step. The cost of building a database improves the effectiveness of high-throughput database construction technology applied to medical clinical testing programs (such as non-invasive prenatal testing, free tumor DNA gene testing, etc.).

Referring to FIG. 3 and FIG. 4, according to a third embodiment of the present invention, the specific steps of the library construction method based on the break-end repair-plus A one-step method are as follows:

1. Use a non-limiting DNA endonuclease (such as Dnase I, Vvn, or ColE7, etc.) to form a nick or gap in DNA, while using DNA polymerase for nick translation or strand displacement and end repair, and at the 3' end of DNA Add an A base (shown in number 1 in Figure 3 and Figure 4).

Non-limiting endonuclease enzymes are a class of endonucleases that do not have a specific recognition site and can randomly (or preferentially) create nicks or gaps in the DNA duplex. DNA polymerase can recognize nicks or gaps, perform nick translation or strand displacement, and the double strands extend along the 5'-3' direction. When the gaps on the double strands are translated to similar or identical sites, the DNA is disconnected from it. In order to achieve the purpose of fragmenting DNA. At the same time, the polymerization and exonuclease activity of the polymerase can complement the 3' end of the DNA and cleave the 5' end, thereby repairing the ends of the DNA. One-step enzymatic reaction can complete three processes of interruption, end repair and A addition at the same time, saving construction time and saving construction cost.

2. Add a linker at both ends of the DNA fragment, and join different platform connectors for different sequencing platforms. For the CG platform, a bubbling connector with or without U bases (shown as number 2 in Figure 3) can be used; for the Illumina platform, a Y-junction can be used.

As shown in Figure 3, for the CG platform, the following steps are divided into two aspects: PCR amplification or USER enzyme treatment; and single-chain cyclization.

3. PCR amplification: For the PCR strategy, a ligation product containing a bubble linker without U is used, and a nucleic acid single strand complementary to both ends of the target linker sequence is added as a primer for PCR amplification (shown in number 3 in Fig. 3): The DNA double strands are separated by high temperature, and then the primers are bound to the corresponding single strands by cooling, and then extended to obtain a large amount of DNA product.

4, USER enzyme treatment: for the PCR-free strategy, using the U-bubble junction of the ligation product, using U-acting enzymes (such as: USER, or UDG / APE enzymes, etc.) to treat the joints, so that the joints can be connected End (shown in number 4 in Figure 3).

5. Using thermal denaturation, PCR amplification or USER enzyme treatment to obtain a DNA product into a single-stranded deoxynucleic acid, and adding a single-stranded nucleic acid complementary to the head-to-tail sequence (which can be called a mediator fragment) and denaturing single-strand deoxygenation Nucleic acid hybridization and ligation reactions, cyclization of the single-stranded nucleic acid of interest, digestion of the mediated fragment and uncircularized single-stranded nucleic acid with an exonuclease to obtain a single-stranded circular nucleic acid product with a linker (No. 5 in Figure 3) Shown). The product was subjected to rolling circle replication to form a sequencing template product DNB (DNA Nanoball, nucleic acid nanosphere) for subsequent sequencing reactions.

As shown in Fig. 4, for the Illumina platform: PCR amplification was carried out by using a ligation product as a template and adding a nucleic acid single strand complementary to both ends of the desired linker sequence as a primer (shown in No. 3 in Fig. 4). The DNA double strands are separated by high temperature, and after the primers are bound to the corresponding single strands, they are extended to obtain a large amount of DNA product, which can be used for sequencing after bridge PCR.

Compared with the existing library preparation method, the above method of the present invention can perform enzyme disruption and DNA end repair, and add A base in one step. Furthermore, in the case of a bubbling linker with a U base, a PCR-free strategy based on a library of enzyme digestion methods can be achieved. Specifically, the above embodiment subtly binds a non-limiting DNA endonuclease to a DNA polymerase, and uses a 5'-3 DNA polymerase when a non-limiting DNA endonuclease forms a nick on double-stranded DNA. 'Polymerase activity undergoes nick translation or strand displacement to rapidly repair DNA into intact double-stranded DNA fragments, thereby eliminating the effects of non-limiting DNA endonuclease base preference. Moreover, this reaction does not require the support of special equipment, and the temperature control is simple, which can fully meet the needs of automated production. At the same time, the loss of DNA amount can be reduced by reducing the number of manipulation and purification steps, thereby meeting the needs of low initial volume library construction. In addition, using the PCR-free method can not only reduce the requirements for temperature control equipment, but also reduce the base bias and amplification error rate caused by DNA polymerase, and improve the quality of sequencing.

Referring to FIG. 3 and FIG. 4, the present invention also provides a fourth embodiment, which differs from the third embodiment only in that an interrupting enzyme (such as NEB Next dsDNA fragmentase) is substituted for non-limiting DNA incision. Enzyme.

The DNA fragment was randomly disrupted using a disruption enzyme to obtain a small DNA fragment having a 5'-P, 3'-OH overhanging end. At the same time, the polymerization and exonuclease activity of the DNA polymerase in the system can complement the 3'-end of the DNA, cleave the 5'-end, and then increase the reaction temperature, using a DNA polymerase such as Taq at the 5'-end. Add A base. So far, in one step of the enzyme reaction, three steps of interrupting, repairing and adding dATP reaction are simultaneously completed. At the same time, the other enzymes in the reaction are inactivated, and the next reaction can be directly carried out, thereby saving the construction time and saving the construction cost.

The fourth embodiment of the present invention uses a disruption enzyme to randomly generate a nick, and then cleaves the cleavage to form a dsDNA cleavage, and the use of the DNA polymerase can simultaneously perform DNA end repair and add dATP, thereby saving time and cost. Moreover, this reaction does not require the support of other equipment, and the temperature control is simple, and can fully meet the requirements of automated production. At the same time, the loss of DNA amount can be reduced by reducing the number of manipulation and purification steps, thereby meeting the needs of low initial volume library construction. In addition, the PCR-free method not only reduces the requirements for temperature control equipment, but also reduces base unevenness caused by DNA polymerase and improves sequencing quality.

In the third embodiment and the fourth embodiment, the DNA polymerase may be a combination of a Klenow large fragment and Taq DNA polymerase or a combination of DNA polymerase I and Taq DNA polymerase.

The technical solutions of the present invention are described in detail below by way of examples. It should be understood that the embodiments are only illustrative and are not to be construed as limiting the scope of the invention.

The method is applicable to different types of samples, and may be genomically interrupted samples, such as human genomic DNA, E. coli DNA, cerebrospinal fluid DNA containing pathogens, or naturally fragmented DNA, for example, as plasma free DNA (cell-free) DNA, cfDNA). The method is applicable to samples with different initial input amounts, and the sample after fragmentation can be quantified, and the input amount can be 2 ng-100 ng.

Example 1: Library construction of 8 plasma samples

1. Sample collection and processing:

2 ml of venous blood, 1600 g, was centrifuged at 4 ° C for 10 minutes to separate the blood cells from the plasma, and the plasma was further centrifuged at 16,000 g for 10 minutes at 4 ° C to further remove residual white blood cells. Plasma DNA was extracted and finally the DNA was dissolved in 40 μL of TE solution.

2. End repair - add adenylate deoxyribonucleic acid:

Configure the system according to the following table:

table 3

10×T4 Polynucleotide Kinase Buffer (Enzematics) 5μL T4 Polynucleotide Kinase (Enzematics) 0.4μL Klenow large fragment (5U/μL) (NEB) 0.4μL Deoxyribonucleic acid mixture (25 mM each) (Enzematics) 0.4μL Taq DNA polymerase (5U/μL) (Takara) 0.4μL Adenyl deoxyribonucleic acid (10 mM) (Enzematics) 1μL T4 DNA polymerase (3U/μL) (Enzymatics) 0.4μL Enzyme-free pure water 2μL total capacity 10μL

10 μL of the reaction solution was added to DNA homogenized to 40 μL, mixed, and incubated at 37 ° C for 10 min; incubated at 72 ° C for 15 min, and cooled to 4 ° C at a rate of 0.1 sec.

3. Connector connection:

The linker sequence used in the present scheme is as follows (the sequence in this example is from the left to the right of the 5' end to the 3' end, ", the modification group is shown, "the modification group shows phosphorylation, and B10 shows 10 The sequence of the base tag.)

Long chain:

/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(B10)CAACTCCTTGGCTCACA (SEQ ID NO: 1);

Short chain: TTGTCTTCCTAAGACCGCGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 2).

The Connector A mixture (10uM) was formulated as follows:

Table 4

Long chain (100uM) 40μL Short chain (100uM) 40μL

Sodium chloride (5M) 4μL Tris (hydroxymethylaminomethane-hydrochloric acid (1M, pH 7.8) 4μL Disodium edetate (2 mM) 20μL Enzyme-free pure water 292μL total capacity 400.0 μL

Add 2 μL of the prepared adapter mix (10 uM) to the product of the previous step and mix thoroughly.

Configure the system according to the following table:

table 5

Adenosine triphosphate (100 mM) 0.8μL Ligase (600U/μL) (Enzematics) 0.5μL 10×T4 Polynucleotide Kinase Buffer (Enzematics) 3μL Polyethylene glycol 8000 (50%) 12μL Enzyme-free pure water 11.7μL total capacity 28μL

The reaction mixture was mixed with a mixture of the linker and the product, and incubated at 23 ° C for 30 min. After the reaction, 40 μL of Ampure XP magnetic beads (Beckman) was added for suction purification, and 42 μL of TE buffer was dissolved to recover the product. (There are various ways of purifying the reaction product, such as magnetic bead method, column purification method, gel recovery method, etc. Both can be used for replacement. This embodiment is purified by magnetic beads method unless otherwise specified.)

4.PCR (polymerase chain reaction)

The primer sequences are as follows: (The sequence in this example is from the left to the right from the 5' end to the 3' end, ", the sequence shows a modifying group, and the "modification group, which shows phosphorylation."

Primer 1 sequence: /5Phos / GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TTGGAGCCAAGAGGTTG (SEQ ID NO: 4).

Prepare the system according to the following table:

Table 6

10×pfx buffer (Thermo Science) 10μL Deoxyribonucleic acid buffer (25 mM) (Enzematics) 1.6μL Magnesium sulfate (50 mM) (Thermo Science) 4μL Primer 1 (25uM) 2.5μL Primer 2 (25uM) 2.5μL Pfx DNA polymerase (2.5 U/μL) (Thermo Science) 2μL Enzyme-free pure water 37.4μL total capacity 60μL

The above product was recovered in 40 μL of the above step, added to the above system, and mixed, and then reacted according to the following conditions:

Table 7

After completion of the reaction, purification was carried out using 100 μL of Ampure XP magnetic beads, and 30 μL of LTE buffer was dissolved to recover the product. 1 μL of the recovered product was taken, and the product concentration was quantified using a Qubit dsDNAHS Assay Kit (Invitrogen) (the results are shown in Table 8). 1 μL was taken for electrophoresis (results shown in Figure 5).

Table 8 shows the total amount of the eight sample PCR products of this example, which is in accordance with the requirement of single-strand cyclization for each sample PCR product of 20 ng or more.

Table 8

library 1 2 3 4 5 6 7 8 Total amount (ng) 514 190.4 212 1130 388 836 582 682

Figure 5 shows the results of electrophoresis of the eight sample PCR products of this example. The product fragment size is about 250 bp, which is consistent with the plasma DNA library fragment size.

5. Single chain cyclization:

Eight amplification products with different tag sequences were each taken at 20 ng for mixing, and configured into a 48 μL system using TE, and 5 μL of 20 uM-mediated fragment was added and mixed. The reaction system was incubated at 95 ° C for 3 min and immediately placed on ice.

Wherein the mediated fragment has a corresponding complementary sequence for ligating the ends of the single strand, the sequence of which is as follows: (the sequence in this embodiment is from 5' to 3' from left to right)

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

Formulation of reaction system 2:

Table 9

10×TA buffer (Enzematics) 6μL Adenosine triphosphate (100 mM) 0.6μL T4 DNA ligase (fast) (600U/μL) (Enzematics) 0.2μL total capacity 6.8μL

The reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 30 min.

6. Sequencing

The constructed single-stranded circular DNA library was used for DNA nanosphere preparation and CG sequencing. The sequencing process is performed in strict accordance with the standard operating procedures of Complete Genomics Inc. for computer operation and data analysis.

Example 2: PCR-Free library construction of 48 plasma samples

1. Sample collection and processing:

2 ml of venous blood, 1600 g, was centrifuged at 4 ° C for 10 minutes to separate the blood cells from the plasma, and the plasma was further centrifuged at 16,000 g for 10 minutes at 4 ° C to further remove residual white blood cells. Plasma DNA was extracted and finally the DNA was dissolved in 40 μL of TE solution.

2. End repair - add adenylate deoxyribonucleic acid:

Configure the system according to the following table:

Table 10

10×T4 Polynucleotide Kinase Buffer (Enzematics) 5μL T4 Polynucleotide Kinase (Enzematics) 0.6μL Deoxyribonucleic acid mixture (25 mM each) (Enzematics) 0.5μL Taq DNA polymerase (5U/μL) (Takara) 0.4μL Adenyl deoxyribonucleic acid (10 mM) (Enzematics) 1μL T4 DNA polymerase (3U/μL) (Enzymatics) 0.6μL Enzyme-free pure water 1.9μL total capacity 10μL

10 μL of the reaction solution was added to DNA homogenized to 40 μL, mixed, and incubated at 37 ° C for 30 min; incubated at 65 ° C for 15 min, and cooled to 4 ° C.

3. Connector connection:

The linker sequence used in the present scheme is as follows (the sequence in this example is from the left to the right of the 5' end to the 3' end, ", the modification group is shown, "the modification group shows phosphorylation, and B10 shows 10 The tag sequence of the base. ideoxyU shows uracil nucleotides)

Long chain:

/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(B10)CAACTCCTTGGCTCACA (SEQ ID NO: 1);

Short chain: AGCCAAGGAGT/ideoxyU/GAACGACATGGCTACGATCCGACTT (SEQ ID NO: 6).

The Connector A mixture (10uM) was formulated as follows:

Table 11

Long chain (100uM) 40μL Short chain (100uM) 40μL Sodium chloride (5M) 4μL

Tris (hydroxymethylaminomethane-hydrochloric acid (1M, pH 7.8) 4μL Disodium edetate (2 mM) 20μL Enzyme-free pure water 292μL total capacity 400μL

2 μL of the prepared adapter mix (10 uM) was added to the product of the previous step and mixed well.

Configure the system according to the following table:

Table 12

Adenosine triphosphate (100 mM) 0.8μL Ligase (600U/μL) (Enzematics) 1μL 10×T4 Polynucleotide Kinase Buffer (Enzematics) 3μL Polyethylene glycol 8000 (50%) 12μL Enzyme-free pure water 11.2μL total capacity 28μL

The reaction mixture was mixed with a mixture of the linker and the product, and incubated at 23 ° C for 30 min. After the reaction, 24 samples with different label sequences were mixed together, and then one volume of Ampure XP magnetic beads (Beckman) was added for purification, and 55 μL of TE buffer was used to dissolve the recovered product. (There are various ways of purifying the reaction product, such as magnetic bead method, column purification method, gel recovery method, etc. Both can be used for replacement. This embodiment is purified by magnetic beads method unless otherwise specified.)

4.User cut

Configure the system according to the following table:

Table 13

10×TA buffer (Enzematics) 6μL User digestion (1U/μL) (NEB) 1μL total capacity 7μL

7 μL of the reaction solution was added to the product of the previous step, mixed, placed in a 37-mix incubation for 15 min; and cooled to 4 °C.

5. Single chain cyclization:

Formulation of Reaction System 1: 5 μL of 20 uM mediated fragment was added to the product of the previous step and mixed. The reaction system was incubated at 95 ° C for 3 min and immediately placed on ice.

Wherein the mediated fragment has a corresponding complementary sequence for ligating the ends of the single strand, the sequence of which is as follows: (the sequence in this embodiment is from 5' to 3' from left to right)

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

Formulation of reaction system 2:

Table 14

Adenosine triphosphate (100 mM) 0.6 μL T4 DNA ligase (fast) (600U/μL) (Enzematics) 0.2 μL total capacity 0.8 μL

The reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 30 min.

6. Sequencing

The constructed single-stranded circular DNA library was used for DNA nonoball preparation and CG sequencing. The sequencing process is performed in strict accordance with the standard operating procedures of Complete Genomics Inc. for computer operation and data analysis.

Table 15 shows the concentration of the PCR-Free library DNA nanospheres of this example, in accordance with the requirements for sequencing on the BGISEQ platform.

Table 15

library PCR-Free 1 PCR-Free 2 Concentration (ng/μL) 61.3 60.1

Fig. 6 shows the results of the PCR-Free library DNA nanosphere electrophoresis of the present example. Most of the samples are in the pores and cannot be electrophoresed, which is consistent with the fact that the DNA nanospheres cannot run out of the pores during polyacrylamide electrophoresis.

Example 3: Library construction of a pulsed ultrasound interrupted genome

1. DNA sample fragmentation:

The inflammatory yellow cell line DNA was 500 ng, subjected to ultrasonication, purified with 1.5-fold Ampure XP magnetic beads (Beckman), and 22 μL of TE buffer was used to dissolve the recovered product.

2. End repair - add dATP:

Configure the system as shown in Table 16 below.

Table 16

Component Dosage 10× T4 Polynucleotide Kinase Buffer (Enzymatics) 5 μL T4 Polynucleotide Kinase (Enzymatics) 0.6 μL Deoxyribonucleic acid mixture (25 mM each) (Enzymatics) 0.4 μL Taq DNA polymerase (5 U/μL) (Takara) 0.4 μL Klenow large fragment (5 U/μL) (NEB) 0.2 μL dATP (100 mM) (Enzymatics) 0.25 μL T4 DNA polymerase (3 U/μL) (Enzymatics) 0.6 μL Enzyme-free pure water 22.55μL total capacity 30 μL

30 μL of the reaction solution was added to a DNA solution homogenized to 20 μL, mixed, incubated at 37 ° C for 30 min; incubated at 65 ° C for 15 min, and cooled to 4 ° C.

3. Connector connection:

The linker sequence used in the present scheme is as follows (the sequence in this example is from the left to the right of the 5' end to the 3' end, "//" indicates a modifying group, "phos" indicates phosphorylation, and B10 indicates 10 bases. Sequence of tags).

Long chain:

/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (B10) CAACTCCTTGGCTCACA (SEQ ID NO: 1).

Short chain: TTGTCTTCCTAAGACCGCGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 2).

The connector mix (10 μM) was formulated as shown in Table 17 below:

Table 17

Component Dosage Long chain (100μM) 40μL Short chain (100μM) 40μL Sodium chloride (5M) 4μL Tris (hydroxymethylaminomethane-hydrochloric acid (1M, pH 7.8) 4μL Disodium edetate (2 mM) 20μL Enzyme-free pure water 292μL total capacity 400.0 μL

5 μL of the prepared adapter mix (10 μM) was added to the product of the previous step and mixed well.

Configure the system as shown in Table 18 below.

Table 18

Component Dosage Adenosine triphosphate (100 mM) 0.8μL Ligase (600U/μL) (Enzymatics) 1μL 10×T4 Polynucleotide Kinase Buffer (Enzymatics) 3μL Polyethylene glycol 8000 (50%) 12μL Enzyme-free pure water 8.2μL total capacity 25μL

The ligation reaction system was mixed with the mixture of the linker and the product, and incubated at 23 ° C for 30 min. After the reaction, 40 μL of Ampure XP magnetic beads (Beckman) was added for purification, and 42 μL of TE buffer was dissolved to recover the product.

4.PCR (polymerase chain reaction)

The primer sequences are as follows (the sequence in this example is from the left end to the 5' end to the 3' end, "//" indicates a modifying group, and "phos" indicates phosphorylation):

Primer 1 sequence: /5Phos / GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TTGGAGCCAAGAGGTTG (SEQ ID NO: 4).

The system was prepared according to the following Table 19.

Table 19

Component Dosage 10×pfx buffer (Thermo Science) 10μL Deoxyribonucleic acid buffer (25 mM) (Enzymatics) 1.6μL Magnesium sulfate (50 mM) (Thermo Science) 4μL Primer 1 (25 μM) 2.5μL Primer 2 (25 μM) 2.5μL Pfx DNA polymerase (2.5 U/μL) (Thermo Science) 2μL Enzyme-free pure water 57.4μL total capacity 80μL

The product was recovered in 40 μL of the above step, added to the above system, and mixed, and then reacted under the conditions shown in Table 20.

Table 20

After completion of the reaction, purification was carried out using 100 μL of Ampure XP magnetic beads, and 30 μL of LTE buffer was dissolved to recover the product. 1 μL of the recovered product was taken, and the product concentration was quantified using a Qubit dsDNA HS Assay Kit (Invitrogen). Carry out the next reaction.

5. Single chain cyclization:

The reaction system 1 was prepared: 320 ng of the amplified product was mixed, and a 65 μL system was placed with TE, and 5 μL of a 20 μM-mediated fragment was added and mixed. The reaction system was incubated at 95 ° C for 3 min and immediately placed on ice.

Wherein the mediated fragment has a corresponding complementary sequence for ligation of both ends of the single strand, the sequence of which is as follows: (the sequence in this example is from the left end to the 5' end to the 3' end).

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

The reaction system 2 of Table 21 was prepared.

Table 21

Component Dosage 10×TA buffer (Enzymatics) 12μL Adenosine triphosphate (100 mM) 1.2μL T4 DNA ligase (fast) (600U/μL) (Enzymatics) 0.4μL Enzyme-free pure water 36.4μL total capacity 50μL

The reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 30 min.

6. Sequencing

The constructed single-stranded circular DNA library was taken for DNA nanosphere preparation and BGISEQ-500/1000 was sequenced. The sequencing process is performed in strict accordance with the standard operating procedures of BGISEQ-500/1000 for computer operation and data analysis.

Table 22 shows the results of PCR and sequencing of a genomic sample, and the PCR yield satisfies the need to interrupt the library. The output of the sequencing data as well as the depth and coverage have met the needs of data analysis.

Table 22

PCR yield (ng) 723 Total number of sequencing fragments 2,072,698,572 Total number of base pairs 92,277,215,100 Alignment rate 89.04% Unique comparison rate 82.94% Evaluation of sequencing depth 26.31x Coverage 99.46%

Figure 7 shows the sequencing depth distribution of a genomic sample. The sequencing depth is consistent with the Poisson distribution, and the sequencing depth is concentrated at about 30x, which is in line with the data requirements of human genome resequencing.

Figure 8 shows the cumulative GC content of a genomic sample. Although the coverage of high GC content fragments is reduced, the coverage is close to 1, indicating that most of the high GC content fragments can be detected, all different GCs. The coverage of the fragment after homogenization is substantially maintained.

Example 4: Library construction of 8 genomes interrupted by NEB interruptase

1. DNA sample fragmentation:

100 ng of inflammatory yellow cell line DNA was purified with 1.2 times Ampure XP magnetic beads (Beckman) (this step does not need to be performed when the DNA is dissolved in EDTA-free solution or enzyme-free pure water), and 18 μL of enzyme-free pure water is used to dissolve the recovered product. .

Configure the system as shown in Table 23 below.

Table 23

Component Dosage 10×NEB interrupted enzyme buffer V2 (NEB) 2μL

NEB interrupt enzyme (NEB) 2μL total capacity 4μL

4 μL of the reaction solution was added to DNA homogenized to 16 μL, mixed, incubated at 4 ° C for 5 min; incubated at 37 ° C for 20 min, incubated at 65 ° C for 15 min, and cooled to 4 ° C. After the reaction, 20 μL of LAmpure XP magnetic beads (Beckman) was added, mixed, and allowed to stand for 5 minutes. The supernatant was taken, and 22 μL of Mempure XP magnetic beads (Beckman) was added to the supernatant for purification, and 42 μL of TE buffer was used to dissolve the recovered product. .

2. End repair - add dATP:

Configure the system as shown in Table 24 below.

Table 24

Component Dosage 10×T4 Polynucleotide Kinase Buffer (Enzymatics) 5μL T4 Polynucleotide Kinase (Enzymatics) 0.8μL Deoxyribonucleic acid mixture (25 mM each) (Enzymatics) 0.4μL Taq DNA polymerase (5 U/μL) (Takara) 0.4μL dATP (100 mM) (Enzymatics) 0.25 μL T4 DNA polymerase (3 U/μL) (Enzymatics) 1μL Enzyme-free pure water 1.97μL total capacity 10μL

10 μL of the reaction solution was added to a DNA solution homogenized to 40 μL, mixed, incubated at 15 ° C for 30 min; incubated at 65 ° C for 15 min, and cooled to 4 ° C.

3. Connector connection:

The linker sequence used in the present scheme is as follows (the sequence in this example is from the left to the right of the 5' end to the 3' end, "//" indicates a modifying group, "phos" indicates phosphorylation, and B10 indicates 10 bases. Sequence of tags).

Long chain:

/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(B10)CAACTCCTTGGCTCACA (SEQ ID NO: 1);

Short chain: TTGTCTTCCTAAGACCGCGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 2).

The joint mix (10 μM) was formulated as shown in Table 25 below.

Table 25

Component Dosage Long chain (100μM) 40μL Short chain (100μM) 40μL Sodium chloride (5M) 4μL Tris (hydroxymethylaminomethane-hydrochloric acid (1M, pH 7.8) 4μL

Disodium edetate (2 mM) 20μL Enzyme-free pure water 292μL total capacity 400.0 μL

2 μL of the prepared adapter mix (10 μM) was added to the product of the previous step and mixed well.

Configure the system as shown in Table 26 below.

Table 26

Component Dosage Adenosine triphosphate (100 mM) 0.8μL Ligase (600U/μL) (Enzymatics) 1μL 10×T4 Polynucleotide Kinase Buffer (Enzymatics) 3μL Polyethylene glycol 8000 (50%) 12μL Enzyme-free pure water 12.2μL total capacity 29μL

The ligation reaction system was mixed with the mixture of the linker and the product, and incubated at 23 ° C for 30 min. After the reaction, 40 μL of Ampure XP magnetic beads (Beckman) was added for purification, and 22 μL of TE buffer was dissolved to recover the product.

4.PCR (polymerase chain reaction)

The primer sequences are as follows: (The sequence in this example is from the left end to the 5' end to the 3' end, "//" indicates a modifying group, and "phos" indicates phosphorylation).

Primer 1 sequence: /5Phos / GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TTGGAGCCAAGAGGTTG (SEQ ID NO: 4).

The system was prepared according to the following Table 27.

Table 27

Component Dosage 10×pfx buffer (Thermo Science) 10μL Deoxyribonucleic acid buffer (25 mM) (Enzymatics) 1.6μL Magnesium sulfate (50 mM) (Thermo Science) 4μL Primer 1 (25 μM) 2.5μL Primer 2 (25 μM) 2.5μL Pfx DNA polymerase (2.5 U/μL) (Thermo Science) 2μL Enzyme-free pure water 67.4 μL total capacity 90μL

10 μL of the recovered product was taken in the above step, added to the above system, and mixed, and the reaction was carried out under the conditions of Table 28 below.

Table 28

After completion of the reaction, purification was carried out using 100 μL of Ampure XP magnetic beads, and 22 μL of LTE buffer was dissolved to recover the product. 1 μL of the recovered product was taken, and the product concentration was quantified using a Qubit dsDNA HS Assay Kit (Invitrogen). Carry out the next reaction.

5. Single chain cyclization:

Preparation of Reaction System 1: Eight ng amplification products with different tag sequences were each mixed for 20 ng, configured into 48 μL system with TE, and 5 μL of 20 μM mediated fragment was added and mixed. The reaction system was incubated at 95 ° C for 3 min and immediately placed on ice.

Wherein the mediated fragment has a corresponding complementary sequence for ligation of both ends of the single strand, the sequence of which is as follows: (the sequence in this example is from the left end to the 5' end to the 3' end).

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

The reaction system 2 of Table 29 was prepared.

Table 29

Component Dosage 10×TA buffer (Enzymatics) 6μL Adenosine triphosphate (100 mM) 0.6μL T4DNA ligase (fast) (600U/μL) (Enzymatics) 0.2μL total capacity 6.8μL

The reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 30 min.

6. Sequencing

The constructed single-stranded circular DNA library was taken for DNA nanosphere preparation and BGISEQ-500/1000 was sequenced. The sequencing process is performed in strict accordance with the standard operating procedures of BGISEQ-500/1000 for computer operation and data analysis.

Table 30 shows the PCR yields of 8 enzyme-interrupted genomic samples, and the yields of the 8 samples all met the requirements of the BGISEQ-500/1000 sequencing platform.

Table 30

sample Concentration (ng/μl) Total amount (ng) 1 5.96 119.2

2 6.25 125 3 5.68 113.6 4 6.41 128.2 5 6.78 135.6 6 5.87 117.4 7 6.69 133.8 8 6.4 128

Figure 9 shows the electrophoresis pattern of PCR products of 8 enzyme-interrupted genomic samples. The fragment size of the PCR product is about 250 bp, which meets the requirements of the BGISEQ-500/1000 sequencing platform.

Example 5: Breaking-End Repair Based on CG Platform - Adding A One-Step Method to Construct Library

1. Interruption - end repair - plus adenylate deoxyribonucleic acid:

The DNA samples were homogenized to 10 ng/μL. Configure the system according to Table 31 below:

Table 31

Component Dosage 10×NEB interrupted enzyme buffer V2 (NEB) 3μL Magnesium chloride 200 mM) (NEB Corporation) 1.5μL Klenow large fragment (5U/μL) (NEB) 1μL Deoxyribonucleic acid mixture (25 mM each) (Enzematics) 0.12μL Taq DNA polymerase (5U/μL) (Takara) 0.5μL Adenyl deoxyribonucleic acid (100 mM) (Enzematics) 0.15μL NEB interrupt enzyme (NEB) 2μL Enzyme-free pure water 11.88μL total capacity 20μL

20 μL of the reaction solution was added to the homogenized DNA, mixed, incubated at 37 ° C for 15 min; and incubated at 65 ° C for 15 min.

This step can also use the system of Table 32 below:

Table 32

Component Dosage 10×NEB interrupted enzyme buffer V2 (NEB) 3μL Magnesium chloride 200 mM 1.5μL DNA polymerase I (10U/μL) (NEB) 1μL Deoxyribonucleic acid mixture (25 mM each) (Enzematics) 0.12μL Taq DNA polymerase (5U/μL) (Takara) 1μL Adenyl deoxyribonucleic acid (100 mM) (Enzematics) 0.15μL NEB Interruption Enzyme (NEB) (NEB) 2μL

Enzyme-free pure water 11.88μL total capacity 20μL

10 μL of the reaction solution was added to the homogenized DNA, mixed, incubated at 37 ° C for 15 min; and incubated at 65 ° C for 15 min.

2. Connector connection:

The linker sequence used in the present scheme is as follows (the sequence in this example is from the left to the right of the 5' end to the 3' end, "//" indicates a modifying group, "phos" indicates phosphorylation, and the font bold indicates a tag sequence. , * Sulfation modification.)

Long chain 1:

/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATGTCATAAATCAACTCCTTGGCTC*A*C*A (SEQ ID NO: 6);

Long chain 2:

5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATGTCATAAATCAACTCCTTGGCTCACA (SEQ ID NO: 6).

Short chain 1: TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 7);

Short chain 2: TTGTCTTCCTAAGACCGCGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 8);

Short chain 3: AGGAGTTGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 9);

Short chain 4: AGCCAAGGTCAGTAACGACATGGCTACGATCCGACTT (SEQ ID NO: 10).

Mixture A (25 μM) was formulated as shown in Table 33 below:

Table 33

Component Dosage Long chain (one of them, 200μM) 12.5 μL Short chain (one of them, 200μM) 12.5 μL Sodium chloride (5M) 1.2μL Tris (hydroxymethylaminomethane-hydrochloric acid (1M, pH 7.8) 1.2μL Disodium edetate (20 mM) 0.5μL Enzyme-free pure water 72.5μL total capacity 100.0μL

4 μL of the prepared adapter mix (25 μM) was added to the product of the previous step and mixed well.

Configure the system according to the following table 34:

Table 34

Component Dosage Adenosine triphosphate (100 mM) 0.8μL Ligase (600U/μL) (Enzematics) 0.8μL

10×T4 Polynucleotide Kinase Buffer (Enzematics) 5μL Polyethylene glycol 8000 (50%) 16μL Enzyme-free pure water 23.4μL total capacity 46μL

The reaction mixture was mixed with a mixture of the linker and the product, and incubated at 23 ° C for 60 min. After the reaction, purification was carried out with 50 ul of Ampure XP magnetic beads, and the product was dissolved in 46 μL of LTE buffer.

3. Polymerase chain reaction

The primer sequences are as follows (the sequence in this example is from the left end to the 5' end to the 3' end, "//" indicates a modifying group, and "phos" indicates phosphorylation):

Primer 1 sequence: /5Phos / GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TTGGAGCCAAGAGGTTG (SEQ ID NO: 4).

Prepare the system according to the following table 35:

Table 35

Component Dosage 10×pfx buffer (Thermo Science) 10μL Deoxyribonucleic acid buffer (25 mM) (Enzematics) 1.6μL Magnesium sulfate (50 mM) (Thermo Science) 4μL Primer 1 (25 μM) 2.5μL Primer 2 (25 μM) 2.5μL Pfx DNA polymerase (2.5 U/μL) (Thermo Science) 2μL Enzyme-free pure water 57.4μL total capacity 80μL

The above product was recovered in 20 μL of the above step, added to the above system, and mixed, and then reacted according to the conditions of Table 36:

Table 36

After completion of the reaction, purification was carried out using 100 μL of Ampure XP magnetic beads, and 30 μL of LTE buffer was dissolved to recover the product. 1 μL of the recovered product was taken, and the product concentration was quantified using a Qubit dsDNA HS Assay Kit (Invitrogen). Carry out the next reaction.

4. Single chain cyclization:

330 ng of the above DNA was placed in a 65 μL system (Reaction System 1) with TE, and 5 μL of a 25 μM-mediated fragment was added and mixed. The reaction system was incubated at 95 ° C for 3 min; at 4 ° C for 10 min.

Wherein the mediated fragment has a corresponding complementary sequence for joining the two ends of the single strand, the sequence of which is as follows (the sequence in this example is from the left end to the 5' end to the 3' end):

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

Prepare Reaction System 2 (Table 37):

Table 37

Component Dosage 10×TA buffer (Enzematics) 12μL Adenosine triphosphate (100 mM) 1.2μL T4 DNA ligase (fast) (600U/μL) (Enzematics) 0.42μL Enzyme-free pure water 36.38μL total capacity 50μL

The reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 60 min.

5. Digest linear single chain:

Configure the reaction buffers in Table 38 below:

Table 38

Component Dosage 10×TA buffer (Enzematics) 4μL Exonuclease 1 (20 U/μL) (NEB) 1.9μL Exonuclease 3 (100U/μL) (NEB) 1.3μL Enzyme-free pure water 1.5μL total capacity 8μL

8 μL of the reaction buffer was added to 120 μL of the reaction product of the previous step, mixed, and incubated at 37 ° C for 30 min. 6 μL of ethylenediaminetetraacetic acid (500 mM) was added and mixed. Purification was carried out using 170 μL of PEG32 magnetic beads, and 50 μL of TE buffer was used to dissolve the product.

Figure 10 shows the bio-analysis 2100 results of PCR products of two enzyme-interrupted genomic samples. The fragment size of the PCR product is about 250 bp, which meets the requirements of the BGISEQ-500/1000 sequencing platform.

Example 6: Breaking-End Repair Based on Illumina Sequencing Platform - Adding A One-Step Method to Build a Library

1. Interruption - end repair - plus adenylate deoxyribonucleic acid:

The DNA samples were homogenized to 10 ng/μL. Configure the system according to the following table 39:

Table 39

Component Dosage 10×fragmentation buffer (NEB) 3μL Bovine serum albumin (100mg/mL) (NEB) 0.3μL DNA polymerase I (5U/μL) (NEB Corporation) 1μL Deoxyribonucleic acid mixture (10 mM each) (Enzematics) 0.5μL Taq DNA polymerase (5U/μL) (Takara) 0.5μL Adenyl deoxyribonucleic acid (10 mM) (Enzematics) 1μL Vvn non-restricted endonuclease (1.3U/μL) (NEB) 1μL Enzyme-free pure water 12.7μL total capacity 20μL

20 μL of the reaction solution was added to the homogenized DNA, mixed, incubated at 37 ° C for 15 min; and incubated at 70 ° C for 15 min.

2. Connector connection:

Configure the system according to the following table 40:

Table 40

Component Dosage Ligase mixture (NEB) 15μL NEBNext Illumina Connector (NEB Corporation) 2.5μL Connection enhancer (NEB) 1μL Enzyme-free pure water 35μL total capacity 53.5 μL

The ligation reaction system was mixed with the mixture of the linker and the product, and incubated at 25 ° C for 15 min. After the reaction, 3 μL of USER enzyme (NEB) was added and incubated at 37 ° C for 15 min. After completion of the reaction, 13.5 μL of enzyme-free pure water was added to the reaction system, 55 μL of Ampure XP magnetic beads were added for purification, and 15 μL of LTE buffer was dissolved to recover the product.

3. Polymerase chain reaction

Prepare the system according to the following table 41:

Table 41

Component Dosage NEBNext Q5 Hot Start PCR Mix (NEB) 25μL I7 Primer (NEB Company) 5μL I5 primer (NEB company) 5μL total capacity 35μL

15 μL of the product was recovered in the above step, added to the above system, and mixed, and then reacted according to the conditions of Table 42:

Table 42

After completion of the reaction, purification was carried out using 45 μL of Ampure XP magnetic beads, and 33 μL of LTE buffer was dissolved to recover the product. 1 μL of the recovered product was taken, and the product concentration was quantified using a Qubit dsDNAHS Assay Kit (Invitrogen).

Example 7: Breaking-End Repair Based on CG Platform - Adding A One-Step Method to Build a Library

1. Interruption - end repair - plus adenylate deoxyribonucleic acid:

The DNA samples were homogenized to 10 ng/μL. Configure the system according to the following table 43:

Table 43

Component Dosage 10×fragmentation buffer V2 (NEB) 3μL Klenow large fragment (5U/μL) (NEB) 0.5μL Deoxyribonucleic acid mixture (1 mM each) (Enzematics) 1.2μL Taq DNA polymerase (5U/μL) (Takara) 0.5μL Adenyl deoxyribonucleic acid (5 mM) (Enzematics) 1.8μL Interrupted enzyme (NEB) 3μL Enzyme-free pure water 10μL total capacity 20μL

20 μL of the reaction solution was added to the homogenized DNA, mixed, incubated at 4 ° C for 5 min; incubated at 37 ° C for 10 min; and incubated at 65 ° C for 15 min.

This step can also use the system of Table 44 below:

Table 44

Component Dosage 10×fragmentation buffer (NEB) 3μL Klenow large fragment (5U/μL) (NEB) 0.5μL Deoxyribonucleic acid mixture (1 mM each) (Enzematics) 1.2μL Taq DNA polymerase (5U/μL) (Takara) 0.5μL

Adenyl deoxyribonucleic acid (5 mM) (Enzematics) 1.8μL Interrupted enzyme (NEB) 2μL MgCl ₂ (200 mM) (NEB Corporation) 1.5μL Enzyme-free pure water 9.5μL total capacity 20μL

This step can also use the system of Table 45 below:

Table 45

Component Dosage 10×fragmentation buffer V2 (NEB) 3μL DNA PolI (10U/μL) (NEB Corporation) 0.5μL Deoxyribonucleic acid mixture (1 mM each) (Enzematics) 1.2μL Taq DNA polymerase (5U/μL) (Takara) 0.5μL Adenyl deoxyribonucleic acid (5 mM) (Enzematics) 1.8μL Interrupted enzyme (NEB) 3μL Enzyme-free pure water 9.5μL total capacity 20μL

20 μL of the reaction solution was added to the homogenized DNA, mixed, incubated at 4 ° C for 5 min; incubated at 37 ° C for 20 min; and incubated at 65 ° C for 15 min.

2. Connector connection:

The linker sequence used in the present scheme is as follows (the sequence in this example is from the left to the right of the 5' end to the 3' end, "//" indicates a modifying group, "phos" indicates phosphorylation, and the font bold indicates a tag sequence. , *sulfation modification):

Long chain 1:

/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATGTCATAAATCAACTCCTTGGCTC*A*C*A (SEQ ID NO: 6);

Long chain 2:

5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATGTCATAAATCAACTCCTTGGCTCACA (SEQ ID NO: 6).

Short chain 1: TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 7);

Short chain 2: TTGTCTTCCTAAGACCGCGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 8);

Short chain 3: AGGAGTTGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 9);

Short chain 4: AGCCAAGGTCAGTAACGACATGGCTACGATCCGACTT (SEQ ID NO: 10).

Mixture A (25 μM) was formulated as shown in Table 46 below:

Table 46

Component Dosage Long chain (one of them, 200μM) 12.5 μL Short chain (one of them, 200μM) 12.5 μL Sodium chloride (5M) 1.2μL Tris (hydroxymethylaminomethane-hydrochloric acid (1M, pH 7.8) 1.2μL Disodium edetate (20 mM) 0.5μL Enzyme-free pure water 72.5μL total capacity 100.0μL

4 μL of the prepared adapter mix (25 μM) was added to the product of the previous step and mixed well.

Configure the system according to the following table 47:

Table 47

Component Dosage Adenosine triphosphate (100 mM) 0.8μL Ligase (600U/μL) (Enzematics) 1.2μL 10×T4 Polynucleotide Kinase Buffer (Enzematics) 5μL Polyethylene glycol 8000 (50%) 12μL Enzyme-free pure water 27μL total capacity 46μL

The ligation reaction system was mixed with the mixture of the linker and the product, and incubated at 23 ° C for 30 min. After the reaction, 20 μL of 1×TE was added, purified with 50 μL of Axygen magnetic beads, and the recovered product was dissolved in 46 μL of LTE buffer.

3. Polymerase chain reaction

The primer sequences are as follows (the sequence in this example is from the left end to the 5' end to the 3' end, "//" indicates a modifying group, and "phos" indicates phosphorylation):

Primer 1 sequence: /5Phos / GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TTGGAGCCAAGAGGTTG (SEQ ID NO: 4).

Prepare the system according to the following Table 48:

Table 48

Component Dosage 10×pfx buffer (Thermo Science) 10μL Deoxyribonucleic acid buffer (25 mM) (Enzematics) 1.6μL Magnesium sulfate (50 mM) (Thermo Science) 4μL Primer 1 (25 μM) 2.5μL Primer 2 (25 μM) 2.5μL Pfx DNA polymerase (2.5 U/μL) (Thermo Science) 2μL

Enzyme-free pure water 57.4μL total capacity 80μL

20 μL of the product was recovered in the above step, added to the above system, and mixed, and then reacted according to the conditions of Table 49:

Table 49

After completion of the reaction, purification was carried out using 100 μL of Ampure XP magnetic beads, and 30 μL of LTE buffer was dissolved to recover the product. 1 μL of the recovered product was taken, and the product concentration was quantified using a Qubit dsDNAHS Assay Kit (Invitrogen). Carry out the next reaction.

4. Single chain cyclization:

330 ng of the above DNA was placed in a 65 μL system (Reaction System 1) with TE, and 5 μL of a 25 μM-mediated fragment was added and mixed. The reaction system was incubated at 95 ° C for 3 min; at 4 ° C for 10 min.

Wherein the mediated fragment has a corresponding complementary sequence for joining the two ends of the single strand, the sequence of which is as follows (the sequence in this example is from the left end to the 5' end to the 3' end):

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

The reaction system 2 was prepared according to the following Table 50:

Table 50

Component Dosage 10×TA buffer (Enzematics) 12μL Adenosine triphosphate (100 mM) 1.2μL T4 DNA ligase (fast) (600U/μL) (Enzematics) 0.42μL Enzyme-free pure water 36.38μL total capacity 50μL

The reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 60 min.

5. Digest linear single chain:

Configure the reaction buffers in Table 51 below:

Table 51

Component Dosage

10×TA buffer (Enzematics) 0.8μL Exonuclease 1 (20 U/μL) (NEB) 3.9μL Exonuclease 3 (100U/μL) (NEB) 1.3μL Enzyme-free pure water 2μL total capacity 8μL

8 μL of the reaction buffer was added to 120 μL of the reaction product of the previous step, mixed, and incubated at 37 ° C for 30 min. 6 μL of ethylenediaminetetraacetic acid (500 mM) was added and mixed. Purification was carried out using 170 μL of PEG32 magnetic beads, and 50 μL of TE buffer was used to dissolve the product.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (21)

A method for DNA end repair and addition of A, characterized in that the method comprises: in the same reaction system, using a polymerase to fill or squash the ends of the fragmented DNA in the same reaction system, using a polynuclear core Glycosidase converts a 5' hydroxyl group to a 5' phosphate group and a 3' phosphate group to a 3' hydroxyl group; in the presence of excess dATP, a double stranded polymerase with no 3'-5' exo-activity DATP was added to the 3' end of the DNA. A library construction method based on DNA end repair and addition of A, characterized in that the method comprises the following steps: In the same reaction system, in the presence of dNTP, the ends of the fragmented DNA are filled or flattened by a polymerase, and the 5' hydroxyl group is converted into a 5' phosphate group by a polynucleotide kinase and the 3' phosphate group is used. The group is converted to a 3' hydroxyl group; in the presence of excess dATP, a dATP is added to the 3' end of the double stranded DNA using a polymerase having no 3'-5' exo-activity; In the reaction system, a linker and a ligation reaction mixture were directly added, and the 3'A overhanging double-stranded DNA produced in the previous step was ligated to the linker. The library construction method according to claim 2, wherein the linker is a 3'T protruding joint. The library construction method according to claim 3, wherein the linker is a 3'T protruding bubble type linker; the method further comprising: performing PCR amplification and thermal denaturation and single-stranded nucleic acid cyclization; The linker is a U-containing bubbling type linker; the method further comprises: adding USER enzyme digestion and thermal denaturation and single-stranded nucleic acid cyclization. The library construction method according to claim 3, wherein the linker is a 3'T overhang Y-type linker; the method further comprising: performing PCR amplification and purification of the PCR amplification product; or The linker is a Y-type linker comprising a tag sequence; the method further comprising: purifying the linker ligation product. The library construction method according to claim 3, wherein the linker is a 3'T protruding neck ring type joint. The method according to any one of claims 1 to 6, wherein the polymerase is a T4 DNA polymerase and/or a Klenow large fragment; the polynucleotide kinase is a T4 polynucleotide kinase; The polymerase that does not have 3'-5' exo-activity is Taq polymerase and/or Klenow fragment (3'→5'exo-). The method according to claim 7, wherein the fragmented DNA is physically interrupted DNA, and is contained in every 50 μL of the system, physically interrupting DNA 1-100 ng, T4 DNA polymerase 1.2-10 U, and Klenow large fragment. 0-2U, T4 polynucleotide kinase 4-16U, Taq polymerase 1-4U, each dNTP 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg ion 8-15 mM; conditions of the reaction The reaction was carried out at 15-37 ° C for 10-30 min, then at 65-75 ° C for 10-30 min. The method of claim 7 wherein said fragmented DNA is plasma free DNA or enzymatic cleavage of DNA, contained in every 50 μL of the system, plasma free DNA or enzyme digestion interrupts DNA 1-100 ng, T4 DNA polymerase 0-3 U, Klenow large fragment 0-2 U, T4 polynucleotide kinase 4- 10U, Taq polymerase 1-2U, each dNTP 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg ion 8-10 mM, provided that the content of T4 DNA polymerase and Klenow large fragment is 0; The reaction conditions are 15-37 ° C for 10-30 min, then 65-75 ° C for 10-30 min. A method of DNA disruption-end repair-addition A, characterized in that the method comprises: forming a slit or a gap in DNA by using a non-limiting DNA endonuclease in the same reaction system in the presence of dNTP, Gap cleavage or strand displacement is performed using DNA polymerase and end-repairing; dATP is added to the 3' end of double-stranded DNA using DNA polymerase in the presence of excess dATP. A method of DNA disruption-end repair-addition A, characterized in that the method comprises: randomly interrupting a DNA fragment by using an interrupting enzyme in the same reaction system, and simultaneously using a DNA polymerase for end Repair; end-added dATP reaction using DNA polymerase in the presence of excess dATP. A library construction method based on DNA break-end repair-plus A, characterized in that the method comprises the following steps: In the same reaction system, in the presence of dNTP, a non-restricted endonuclease is used to form a nick or gap in the DNA, while DNA polymerase is used for nick translation or strand displacement and end repair; in the presence of excess dATP, use DNA polymerase adds dATP to the 3' end of double-stranded DNA; In the reaction system, a linker and a ligation reaction mixture are directly added, and the double-stranded DNA produced in the previous step is linked to the linker. A library construction method based on DNA break-end repair-plus A, characterized in that the method comprises the following steps: In the same reaction system, in the presence of dNTP, the DNA fragment was randomly interrupted by the disrupting enzyme, and the DNA polymerase was used for terminal repair; in the presence of excess dATP, the DNA polymerase was used for the terminal dATP reaction; In the reaction system, a linker and a ligation reaction mixture are directly added, and the double-stranded DNA produced in the previous step is linked to the linker. The library construction method according to claim 12 or 13, wherein the linker is a 3'T protruding bubbling type linker; the method further comprising: performing PCR amplification and thermal denaturation and single-stranded nucleic acid cyclization; or The linker is a U-containing bubbling type linker; the method further comprises: adding USER enzyme digestion and thermal denaturation and single-stranded nucleic acid cyclization. The library construction method according to claim 12 or 13, wherein the linker is a 3'T overhang Y-type linker; the method further comprises: performing PCR amplification and purification of the PCR amplification product. The library construction method according to claim 12 or 13, wherein the linker is a 3'T protruding neck ring type joint. The method according to any one of claims 10 to 13, wherein the DNA polymerase is a combination of a large Klenow fragment and Taq DNA polymerase, or a combination of DNA polymerase I and Taq DNA polymerase, or T4 DNA. A combination of polymerase and Taq DNA polymerase. A reaction system for DNA end-repair and addition of A, characterized in that the system is contained in every 50 μL of the system, physically interrupting DNA 1-100 ng, T4 DNA polymerase 1.2-10 U, Klenow large fragment 0-2 U, T4 polynucleotide kinase 4-16U, Taq polymerase 1-4U, each dNTP 0.02-0.2 mM, additional dATP 0.4-1 mM, and Mg ions 8-15 mM; or The system contains DNA 1-100 ng, T4 DNA polymerase 0-3U, Klenow large fragment 0-2U, T4 polynucleotide kinase 4-10U, Taq, contained in every 50 μL of the system, plasma free DNA or restriction enzyme digestion. The polymerase is 1-2 U, each dNTP is 0.02-0.2 mM, the additional dATP is 0.4-1 mM, and the Mg ion is 8-10 mM, provided that the content of the T4 DNA polymerase and the Klenow large fragment are different. A kit for DNA end-repair and addition of A, characterized in that the kit is polymerized according to T4 DNA polymerase 1.2-10 U, Klenow large fragment 0-2 U, T4 polynucleotide kinase 4-16 U, Taq polymerization. Enzyme 1-4U, each 1-10nmol of each dNTP, additional dATP 20-50nmol, and Mg ion 400-750nmol, forming a kit unit for dilution into a 50μL reaction system; or The kit is based on T4 DNA polymerase 0-3U, Klenow large fragment 0-2U, T4 polynucleotide kinase 4-10U, Taq polymerase 1-2U, each dNTP each 1-10 nmol, additional dATP 20- 50 nmol, and Mg ion 400-500 nmol, provided that the content of the T4 DNA polymerase and the large Klenow fragment are different, forming a kit unit for dilution into a 50 μL reaction system. A reaction system for DNA disruption-end repair-addition A, characterized in that the system is contained in every 30 μL of the system, 5-100 ng of genomic DNA, 0.5-3 U of NEB interrupting enzyme, and has 3'-5' Exo-active polymerase 0-20U or/and polymerase 0-20U with strand displacement activity, polymerase 1-15U without 3'-5' exo-activity, 0.02-0.2 mM per dNTP, additional dATP 0.4-1 mM, and Mg ions 8-15 mM; or The system contains, in every 30 μL of the system, 5-100 ng of genomic DNA, 0.5-3 U of non-limiting DNA endonuclease, 0-20 U or/and chain displacement activity with 3'-5' exo-activity. The polymerase 0-20 U, polymerase 1-15 U without 3'-5' exo-activity, 0.02-0.2 mM for each dNTP, 0.4-1 mM for additional dATP, and 8-15 mM for Mg ions. A DNA interrupt-end repair-plus A reaction kit, characterized in that the kit is 5-100 ng of genomic DNA, NEB interrupts the enzyme 0.5-3 U, and has a 3'-5' exo-activity polymerization. Enzyme 0-20U or/and polymerase 0-20U with strand displacement activity, polymerase 1-15U without 3'-5' exo-activity, 0.02-0.2 mM for each dNTP, additional dATP 0.4-1 mM, and Mg ion 8-15 mM, Forming a kit unit for dilution into a 30 μL reaction system; or The kit is based on genomic DNA 5-100 ng, non-limiting DNA endonuclease 0.5-3 U, polymerase 0-20 U with 3'-5' exo-activity, and/or polymerase 0-20U with strand displacement activity. , 1-15 U without 3'-5' exo-activity, 0.02-0.2 mM for each dNTP, 0.4-1 mM for additional dATP, and 8-15 mM for Mg ions, forming a kit for dilution into a 30 μL reaction system unit.

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