WO2019006975A1

WO2019006975A1 - In situ whole genome chromatin conformation capture method for infinitesimal cells

Info

Publication number: WO2019006975A1
Application number: PCT/CN2017/114475
Authority: WO
Inventors: 颉伟; 杜振海
Original assignee: 清华大学
Priority date: 2017-07-07
Filing date: 2017-12-04
Publication date: 2019-01-10
Also published as: CN107217309A

Abstract

Provided is a method for constructing a DNA sequencing library of a genome to be tested, the method comprising: (1) digesting a genome to be tested by means of a restriction enzyme; (2) performing biotin labelling on digested products; (3) linking the biotin-labelled products using a DNA ligase; (4) decrosslinking the linked products; (5) purifying the decrosslinked products; (6) performing ultrasonic and precipitation treatments on the purified products, wherein the precipitation treatment is to bring the sonicated products into contact with streptavidin magnetic beads to obtain target DNA fragments bound to streptavidin magnetic beads; and (7) constructing a library based on the target DNA fragments bound to streptavidin magnetic beads.

Description

Minimal amount of cell in situ whole genome chromatin conformation capture

Priority information

The present application claims priority to and the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit.

Technical field

The invention relates to the field of biotechnology. In particular, the invention relates to methods of constructing DNA sequencing libraries of a genome to be tested and uses thereof. More specifically, the present invention relates to a method of constructing a DNA sequencing library of a genome to be tested, a method of determining DNA sequence information of a genome to be tested, and a method of determining a three-dimensional spatial structure of a genome to be tested.

Background technique

In recent years, with the development of genomics, the Human Genome Project has successfully mapped human genomic DNA sequences. The Human Genome Encyclopedia plans to analyze tens of thousands of genes and hundreds of thousands of different gene regulatory elements. Recent studies have shown that the three-dimensional structure of the genome has an important impact on the function of genome expression and regulation. Therefore, how to obtain the three-dimensional structural information of the genome of different cell types in different cell cycles, in different stages of development and differentiation, and in the transition from normal cells to disease cells has become an urgent problem to be solved. The development of chromatin conformation capture technology and second-generation sequencing technology has made the whole genome chromosome conformation capture technology based on second-generation sequencing an important means to study the three-dimensional structure of chromosomes. However, genome-wide chromatin conformation capture technology is extremely limited by the number of cells. If a sufficient amount of DNA for building a database containing enough effective ligation events is not available, it is impossible to obtain efficient and sufficiently high-resolution genomic three-dimensional structural information. of.

Therefore, how to establish a DNA sequencing library of a small number of types of cells that are not easily available and effectively use for DNA sequencing and obtain sufficient high-resolution genomic three-dimensional structure information is a problem to be solved.

Summary of the invention

This application is based on the discovery of the following problems by the inventors:

The traditional Hi-C technology has a large reaction system, and there are many steps of centrifugation, washing, and tube exchange. The selection of fragment size before DNA amplification causes a large amount of DNA loss. Moreover, the selection range of DNA fragments is narrower than the degree of enrichment of DNA fragments by ultrasonic interruption, further reducing the amount of DNA used for second-generation sequencing. The above reasons have led to the fact that Hi-C technology is commonly used to study the commonly available cells of the order of magnitude above 107, which is extremely incapable of obtaining a large number of cell types in the early stages of embryonic development. Based on the discovery of the above traditional technical problems by the inventors, the inventors selected by narrowing the reaction system, changing the mode of cell transfer, and changing the size of the DNA fragment. Scope and time, etc., creatively proposed an improved whole genome DNA sequencing library construction method (in this application, referred to as sisHi-C (small scale in situ Hi-C) technology), which can be used to study the low number of cells Up to 10 precious cell types have brought hope to the establishment of genomic three-dimensional structure and gene expression regulation in the early development of embryos.

In a first aspect of the invention, the invention proposes a method of constructing a DNA sequencing library of a genome to be tested. According to an embodiment of the present invention, the method comprises: (1) digesting the genome to be tested with a restriction endonuclease to obtain a digestion treatment product; and (2) subjecting the digestion treatment product to biotin labeling treatment so that Obtaining a biotin labeling treatment product; (3) linking the biotin labeling treatment product with DNA ligase to obtain a ligation product; (4) de-crosslinking the ligation product; (5) decrosslinking the solution Treating the product for purification treatment; (6) subjecting the purified product to ultrasonication and precipitation treatment by contacting the sonicated product with a streptavidin magnetic bead to obtain a binding enzyme affinity a target DNA fragment of the magnetic beads; and (7) a target DNA fragment based on the magnetic chain bound to the streptavidin magnetic beads. A method of constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention can be used to construct a DNA sequencing library having a cell number as low as 10 cells or a genome amount as low as 50 pg, thereby realizing a non-obtainable, small number of types of cells. Capture of genomic chromatin conformation.

In a second aspect of the invention, the invention proposes a sequencing library. According to an embodiment of the present invention, the sequencing library is obtained by the method of constructing a DNA sequencing library of the genome to be tested as described above. Using sequencing libraries according to embodiments of the present invention for sequencing, it is possible to obtain genomic sequence information with a cell number as low as 10 or a genome amount as low as 50 pg and genomic three-dimensional structure information of sufficiently high resolution.

In a third aspect of the invention, the invention proposes a method of determining DNA sequence information of a genome to be tested. According to an embodiment of the present invention, the method comprises: constructing a DNA sequencing library of a genome to be tested according to the method described above; sequencing the DNA sequencing library to obtain a sequencing result; and determining the site based on the sequencing result Describe the DNA sequence information of the genome. Using the method of determining the DNA sequence information of the genome to be tested according to an embodiment of the present invention, it is possible to obtain genomic sequence information of cells having a cell number as low as 10 or a genome amount as low as 50 pg.

In a fourth aspect of the invention, the invention proposes a method for determining a three-dimensional spatial structure of a genome to be tested. According to an embodiment of the present invention, the method comprises: constructing a DNA sequencing library of a genome to be tested according to the method described above; sequencing the DNA sequencing library to obtain a sequencing result; and determining the site based on the sequencing result The three-dimensional spatial structure information of the detected genome is described. With the method of determining the three-dimensional spatial structure of the genome to be tested according to an embodiment of the present invention, it is possible to obtain a sufficiently high-resolution genomic three-dimensional structure information of cells having a cell number as low as 10 or a genome amount as low as 50 pg.

It should be noted that the genome to be tested proposed by the present invention refers to a whole genome or a partial genome of a cell or a tissue, and the genome is composed of chromatin or chromosome. Those skilled in the art will appreciate that the source of the genome is not particularly limited and can be obtained from any possible route, either directly from a commercial market, directly from other laboratories, or directly from a cell or tissue. Extracted from the sample.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

1 is a flow chart of a method of constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention;

2 is a flow chart of a method of constructing a DNA sequencing library of a genome to be tested according to still another embodiment of the present invention;

3 is a flow chart of a method of constructing a DNA sequencing library of a genome to be tested according to still another embodiment of the present invention;

4 is a flow chart of a TruSeq library according to an embodiment of the present invention;

FIG. 5 is a flowchart of a TruSeq database according to still another embodiment of the present invention; FIG.

6 is a flow chart of a TruSeq library according to still another embodiment of the present invention;

7 is a flow chart of a TruSeq library according to still another embodiment of the present invention;

FIG. 8 is a diagram showing the results of verifying that the method of building a database according to an embodiment of the present invention has significant advantages, according to an embodiment of the present invention.

Detailed ways

The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.

Method for constructing DNA sequencing library of test genome

In a first aspect of the invention, the invention proposes a method of constructing a DNA sequencing library of a genome to be tested. According to an embodiment of the present invention, referring to FIG. 1, the method includes: S100: digesting a genome to be tested with a restriction endonuclease to obtain a digestion treatment product; S200: performing biotin labeling treatment on the digestion treatment product, In order to obtain a biotin labeling treatment product; S300: ligating the biotin labeling treatment product with DNA ligase to obtain a ligation product; S400: de-crosslinking the ligation product; S500: decrosslinking the treated product Purification treatment; S600: subjecting the purified treatment product to ultrasonication and precipitation treatment by contacting the sonicated product with a streptavidin magnetic bead to obtain a streptavidin-coupled magnetic bead Target DNA fragment; and S700: based on a target DNA fragment bound with a streptavidin magnetic beads. A method of constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention can be used to construct a DNA sequencing library having a cell number as low as 10 or a genome amount as low as 50 pg, thereby realizing a readily available, small number of types of cells. Capture of the genomic chromatin conformation.

According to an embodiment of the invention, referring to Figure 2, the genome to be tested is obtained by lysing cells or tissues, optionally the cells are cell lines or primary cells. The cells or tissues are lysed to release the genome in the cells or tissues.

According to an embodiment of the invention, referring to Figure 3, the cells or tissue are previously subjected to a formaldehyde cross-linking treatment. In turn, formaldehyde temporarily immobilizes DNA-protein and protein-protein complexes that are spatially close in the natural state of cells or tissues.

According to an embodiment of the invention, the cell or genome is subjected to a transfer treatment by a mouth pipette. The inventor found that using the mouth The precise transfer of cells or tissue by the pipette can effectively avoid cell loss caused by centrifugation of the replacement solution.

According to an embodiment of the invention, the restriction enzyme is MboI. The inventors found that the recognition site of MboI is GATC, the base distribution is uniform, and there is no obvious preference when cutting on the genome; at the same time, the recognition site of MboI is four bases, compared with the commonly used six The base restriction endonuclease has a higher cutting frequency and a higher resolution of the theoretically obtained data. Finally, MboI is highly commercialized and low in cost, and can be easily obtained, thereby effectively controlling the entire DNA sequencing library. cost.

According to an embodiment of the present invention, the biotin labeling treatment is carried out by treating the digestion treatment product with adenine triphosphate deoxynucleotide, guanine deoxynucleotide triphosphate, thymidine triphosphate deoxynucleotide The biotin-labeled triphosphate cytosine deoxynucleotide and the DNA polymerase large fragment were contacted, and the contact was carried out at 37 ° C for 1.5 hours. Biotin can be efficiently labeled to the end of the MboI fragment by the above method.

According to an embodiment of the invention, the ligation process is a T4 linkage, which is carried out by contacting the ligation product with proteinase K, SDS and sodium chloride. By ligation with T4 DNA ligase, different nick ends of DNA can be ligated into a circular chimeric molecule, and the DNA can be efficiently released by separating the DNA from the bound protein by the above-described decrosslinking treatment.

According to an embodiment of the present invention, the purification treatment is carried out by contacting the decrosslinked treatment product with pre-cooled anhydrous ethanol, which is carried out at -80 ° C for 15 minutes. Using the purification method described above, DNA is efficiently precipitated at the bottom of the tube to further purify the DNA.

According to an embodiment of the invention, the decrosslinking treatment product is contacted with hepatic glycogen and sodium acetate during the purification treatment. The inventors have found that in the process of purifying DNA, since the number of cells is small, the amount of DNA is extremely small, and co-precipitation with hepatic glycogen and DNA can indicate the position of DNA precipitation in the EP tube, thereby effectively preventing DNA loss during washing. .

According to an embodiment of the invention, the ultrasound is performed for 134 s with a Peak Power of 50, a Duty Factor of 20, and a Cycles/Burst of 200. Among them, Peak Power indicates the highest incident power, which is the instantaneous ultrasonic power acting on the sample; Duty Factor indicates the working coefficient, that is, the time when the ultrasonic wave acts on the sample as a percentage of the total time period; Cycles/Burst indicates that the ultrasonic wave acts on the sample during the ultrasonic wave The number of energy transfers. The inventors have found that under the above ultrasonic conditions, DNA loss during DNA fragment size selection can be effectively reduced.

According to an embodiment of the present invention, referring to FIG. 4, the database in S700 is built by TruSeq, and S700 includes S710: end-repairing, S720: end-addition of target DNA fragment bound with streptavidin magnetic beads. Adenine phosphate deoxyribonucleotides and S730: ligation sequencing linker sequences were processed.

According to an embodiment of the present invention, after the S710 end repair treatment, the S720 end plus adenosine triphosphate deoxyribonucleotide treatment further comprises a Tween wash of the magnetic bead treatment; preferably, the S720 end plus adenosine triphosphate deoxyribose After the nucleotide treatment, the S730 is connected to the sequencing linker sequence to further include a Tween wash to treat the magnetic beads; Preferably, the S730-linked sequencing linker sequence further comprises a Tween wash to treat the magnetic beads. After each step of the reaction, the magnetic beads are directly washed twice with the Tween washing solution, and the solution can be changed simply and quickly, thereby avoiding the loss of DNA during the purification process.

According to an embodiment of the present invention, with reference to Figure 5, it is further included that the ligation sequencing linker sequence processing product is subjected to S740: DNA first elution treatment and S750: PCR amplification. After the first elution treatment, the target DNA fragment can be efficiently eluted from the streptavidin magnetic beads, and the target DNA can be efficiently enriched by PCR amplification.

According to an embodiment of the present invention, referring to Figure 6, further comprising S760: combining the PCR amplification product with AMPure XP magnetic beads, optionally, with reference to Figure 7, further comprising S770: PCR to be combined with AMPure XP magnetic beads The amplified product is subjected to a second elution treatment of DNA. Thereby, a DNA fragment having a size ranging from 200 base pairs to 1000 base pairs is obtained.

A sequencing library obtained by the above method according to an embodiment of the present invention can be used as a sisHi-C library for second generation sequencing.

The above method according to an embodiment of the present invention uses a small reaction system to increase the efficiency of enzymatic cleavage and ligation reaction while greatly reducing the cost; accurately transferring cells using a mouth pipette before cell lysis avoids the replacement of the solution by centrifugation Cell loss; binding of DNA to streptavidin magnetic beads for TruSeq library construction, use of milder magnetic bead wash conditions, and controlled library construction in the same EP tube, these measures successfully bypass the purification and recovery steps And effectively reduce the DNA loss during the construction process; optimize the condition of ultrasonic to break the DNA fragment, elute the DNA in two steps, and retain all the DNA in the range of 200bp to 1000bp after PCR, further improve The amount of DNA that is finally available. The whole method not only significantly reduces the amount of starting cells, but also costs one-tenth of the cost of traditional methods, thus efficiently achieving genomic chromatin capture.

It will be understood by those skilled in the art that devices, devices, units, and modules for constructing a DNA sequencing library of a genome to be tested obtained based on the method described in the present application are also within the scope of the present application, the device, device, The advantages of the unit and module are similar to those described above and will not be described in detail here. It will be understood by those skilled in the art that any device known in the art suitable for performing the above operations can be employed as a component of each of the above units. The term "connected" as used herein is used in a broad sense and may be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms may be understood by one of ordinary skill in the art.

Sequencing library

Method for determining DNA sequence information of a genome to be tested

In a third aspect of the invention, the invention proposes a method of determining DNA sequence information of a genome to be tested. Root According to an embodiment of the present invention, the method comprises: constructing a DNA sequencing library of a genome to be tested according to the method described above; sequencing the DNA sequencing library to obtain a sequencing result; and determining the result based on the sequencing result DNA sequence information of the genome to be tested. The method for constructing a DNA sequencing library of the genome to be tested has been described in detail above and will not be described herein. According to an embodiment of the present invention, the method and apparatus for sequencing a sequencing library are not particularly limited, and in view of the maturity of the technique, according to an embodiment of the present invention, second generation sequencing techniques such as SOLEXA, SOLID, and 454 sequencing may be employed. technology. Of course, new sequencing technologies that are being developed or not yet developed, such as single-molecule sequencing technologies such as Helicos' True Single Molecule DNA sequencing technology, Pacific Biosciences' single single molecule, real-time (SMRT.TM.), can also be used. Technology, and nanopore sequencing technology from Oxford Nanopore Technologies, Inc. (Rusk, Nicole (2009-04-01). Cheap Third-Generation Sequencing. Nature Methods 6(4): 244-245).

The inventors have surprisingly found that using a method for determining DNA sequence information of a genome to be tested according to an embodiment of the present invention, it is possible to sensitively, accurately and efficiently determine a genomic or microgenome of a trace amount of cells (starting up to 10 cells). Genomic sequence information with a starting amount of DNA as low as 50 pg).

Method for determining three-dimensional spatial structure of a genome to be tested

In a fourth aspect of the invention, the invention proposes a method for determining a three-dimensional spatial structure of a genome to be tested. According to an embodiment of the present invention, the method comprises: constructing a DNA sequencing library of a genome to be tested according to the method described above; sequencing the DNA sequencing library to obtain a sequencing result; and determining the site based on the sequencing result The three-dimensional spatial structure information of the detected genome is described.

According to a specific embodiment of the present invention, the method comprises constructing a DNA sequencing library of the genome to be tested according to the method described above; performing double-end sequencing on the obtained library to obtain sequence information at both ends of each DNA fragment in the library; The sequence information at both ends is compared to the genome, so that the spatial proximity information between each two segments with different linear distances in the genome can be obtained, and the three-dimensional structure of the genome can be inferred by mathematical methods (Aiden et al.Comprehensive Mapping of long-range interactions reveals folding principle of the human genome. Science. 2009).

The inventors have surprisingly found that the method for determining the sequence information of the chromatin target region of the genome to be tested according to an embodiment of the present invention enables sensitive, accurate and efficient determination of trace cells (up to 10 cells starting from the genome) or trace amounts. A sufficiently high resolution genomic three-dimensional structural information of the genome (initial amount of DNA as low as 50 pg).

The invention is described below with reference to the specific embodiments, which are intended to be illustrative, and are not to be construed as limiting.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. Where the specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., translated by Huang Peitang et al. Subclone Experimental Guide, Third Edition, Science Press) or in accordance with product specifications. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be obtained commercially, for example, from Illumina.

Example 1 Construction of a DNA sequencing library of a cellular genome

1.1 Reagent preparation

Lysis buffer

·10 mM Tris-HCl., pH=7.4

·10mM NaCl

·0.5% NP-40

·0.1mM EDTA

·1X Proteinase Inhibitor

2X biotin and streptavidin magnetic bead binding solution (2X Binding buffer)

·10mM Tris-HCl., pH=8.0

·2M NaCl

·1mM EDTA

Tween wash buffer

· 5mM Tris-HCl., pH=8.0

·1M NaCl

·0.05% Tween

·0.5mM EDTA

1.2 formaldehyde crosslinking

The collected samples were transferred to a freshly prepared PBS solution containing 1% formaldehyde by a mouthpiece under a stereoscopic microscope, fixed at room temperature for 10 minutes, and added with a 2.5 M glycine solution to a final concentration of 0.2 M, and allowed to stand at room temperature for 10 minutes. The sample was transferred to a PBS solution by a mouth pipette and washed once, and then transferred to a PCR tube.

1.3 lysed samples and restriction endonuclease digestion

50 μl of the lysate was added to the PCR tube containing the sample, and the mixture was repeatedly pipetted with a low-absorption filter cartridge and allowed to stand on ice for 50 minutes. Centrifuge to remove the supernatant. Ten microliters of 0.5% SDS solution was added and the metal bath was allowed to stand at 62 ° C for 10 minutes.

25 microliters of water and 10 microliters of a 10% Triton X-100 solution were added to the sample. The mixture was pipetted repeatedly with a pipette, and the sample was placed in a metal bath and incubated at 37 ° C for 15 minutes. Mix the liquid to prevent subsequent SDS from affecting the restriction enzyme activity ring.

Continue to add 5 μl of 10X NEB buffer 2 and 50 U restriction enzyme MboI to the sample, mix and place on a rotary homomixer, and digest at 37 °C for 15 hours to fully digest and separate the chromatin.

1.4 end marking and connection

The next day, the sample was removed from the rotary mixer and placed on a 62 ° C metal bath for 20 minutes to inactivate MboI. On ice, 1 mM adenine deoxynucleotide triphosphate (dATP), 1 mM guanine deoxynucleotide triphosphate (dGTP) and 1 mM thymidine triphosphate deoxynucleotide (dTTP) were added to each sample. Microliters, and 3.75 microliters of biotinylated 0.4 mM cytosine deoxynucleotide (biotin-14-dCTP). After mixing, a large fragment of 10 U DNA polymerase (Klenow) was added, and the sample was placed on a rotary homomixer at 37 ° C for 1.5 hours to label biotin to the end of the MboI fragment.

The solution containing the sample was transferred from the PCR tube to a low adsorption 1.5 ml EP tube. Add 12 μl of 10X NEB T4 DNA ligase reaction solution, 7 μl of 10% Triton X-100, 1.2 μl of 10 mg per microliter of bovine serum albumin, 1 μl of 400 U/ul T4 DNA ligase and 39 μl of water. . After mixing, the sample was placed on a Thermo mixer, 500 rpm, and treated at 24 ° C for 5.5 hours, and the different nick ends were ligated into circular chimeric molecules by DNA ligase.

1.5 decrosslinking and DNA purification

After the completion of the ligation reaction, 100 U of Proteinase K and 12 μl of a 10% SDS solution were added to each sample, and a metal bath at 55 ° C for 30 minutes. Then, 13 μl of 5 M sodium chloride was added, mixed on a vortexer, placed in an oven at 65 ° C, and crosslinked overnight.

On the third day, the sample was taken out of the oven. When the temperature was lowered to room temperature, 1 μl of hepatic glycogen and 15 μl of 3 M sodium acetate were added to each sample, mixed on a vortex, and then 240 μm was added. The pre-cooled anhydrous ethanol was stirred upside down and allowed to stand at -80 ° C for 15 minutes. The precipitate was washed twice with high speed centrifugation with 75% absolute ethanol. After the precipitate was dried, the purified DNA was dissolved in 50 μl of water, and the metal bath was allowed to stand at 37 ° C for 15 minutes. In the process of purifying DNA, since the number of cells is small, the amount of DNA is extremely small, and hepatic glycogen is co-precipitated with DNA to indicate the position of DNA precipitation in the EP tube, preventing DNA loss during washing.

1.6 Ultrasound interrupts DNA and biotin labeling

The sample DNA was shredded into fragments of 300-500 base pairs (bp) using a Covaris M220 ultrasonic DNA disruptor, and the ultrasonic conditions were: Peak Power 50, Duty Factor 20, Cycles/Burst 200, time 134s. The Covaris sonic tube was washed with 20 microliters of water to reduce DNA loss. The inventors have found that DNA loss during DNA fragment size selection can be reduced under the above-described ultrasonic conditions.

Prepare streptavidin magnetic beads (Dynabeads MyOne Streptavidin C1), each sample requires 100 micrograms of magnetic beads. The magnetic beads were washed twice with the Tween washing solution, and then the magnetic beads were resuspended with 70 μl of 2X biotin and streptavidin magnetic bead binding solution, and added to each sample, and pipetted and mixed. uniform. The sample was placed on a rotary mixer and subjected to biotin-labeled precipitation for 50 minutes at room temperature to obtain a labeled DNA fragment of interest.

1.7 Database construction and DNA fragment selection

After biotin-labeled precipitation, the target DNA fragment bearing the biotin label is bound to the streptavidin magnetic beads. Because the binding of biotin-labeled DNA to streptavidin magnetic beads is very stable, these magnetic beads bound to the target DNA can be used directly for TruSeq library construction. Terminal repair was performed in sequence, adenine triphosphate deoxyribonucleotide (dATP) was added to the end of the DNA fragment, and the sequencing sequence was ligated. After each step of the reaction, the magnetic beads are directly washed twice with the Tween washing solution, and the solution can be changed simply and quickly, thereby avoiding the loss of DNA during the purification process.

On the fourth day, after the sequence of the sequencing linker is connected, the sample is placed on a magnetic stand. After the magnetic beads are all adsorbed to the magnetic frame, the solution becomes clear, the supernatant is discarded, and the magnetic beads are washed twice with the Tween washing solution, each time in the Rotate the mixer for 2 minutes at room temperature. Add 20 μl of water to the sample, mix by pipetting, and place the sample on a constant temperature mixer at 66 ° C, 1400 rpm for 20 minutes, and elute the DNA twice. PCR amplification was performed. After the end of the PCR, 72 μl (1:0.48) AMPure XP magnetic beads were added to 150 μl of the sample, pipetted and mixed, and then incubated on a rotary mixer for 5 minutes at room temperature. Transfer the supernatant to a new low-adsorption EP tube, add 78 μl (1:1) AMPure XP magnetic beads, mix and mix for 5 minutes at room temperature on a rotary mixer and discard the supernatant. The magnetic beads were washed twice with 75% absolute ethanol, and after air drying, 50 μl of water was added to the EP tube to elute, thereby obtaining a DNA fragment having a size ranging from 200 base pairs to 1000 base pairs. The library obtained by the above procedure (in the present application, the inventors obtained the library obtained by the above method as a sisHi-C library) can be used for second generation sequencing.

Example 2

In order to compare the effects of the sisHi-C and the previous method (Rao et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014) in a small amount of cell research, the inventors used the mouth. The pipette accurately counts four groups of mouse embryonic stem cells. The number of cells in each group is 500. The inventors then used the sisHi-C and predecessor methods to construct two sets of cells (labeled as Repeat Test 1 and Repeat Test 2, respectively). The concentration of DNA was measured before PCR and the proportion of valid data was analyzed after sequencing of the library. The results in Figure 8A show that the concentration of the two groups of DNA using the sisHi-C method was significantly higher than that of the two groups before the PCR; Figure 8B shows that sisHi-C significantly reduced the loss of DNA in the sequencing data. The exact proportion of PCR-generated sequences is small, and the proportion of valid data is significantly higher than the two groups using the predecessor's method. In summary, compared with the previous methods, the sisHi-C of the present application significantly reduces the loss of DNA during the experiment, very It is suitable for studying the three-dimensional structure of genomes of very few cells.

Moreover, the terms "first" and "second" 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, features defining "first" and "second" may include one or more of the features either explicitly or implicitly. In the description of the present invention, the meaning of "a plurality" is two or more unless specifically and specifically defined otherwise.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, various embodiments or examples described in the specification and features of various embodiments or examples may be combined and combined without departing from the scope of the invention.

Although the embodiments of the present invention have been shown and described, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims

A method for constructing a DNA sequencing library of a genome to be tested, comprising:

(1) digesting the genome to be tested with a restriction endonuclease to obtain a digested product;

(2) subjecting the digestion treatment product to biotin labeling treatment to obtain a biotin labeling treatment product;

(3) ligating the biotin-labeled treatment product with DNA ligase to obtain a ligation product;

(4) subjecting the linked product to decrosslinking treatment;

(5) purifying the cross-linked product;

(6) subjecting the purified product to ultrasonication and precipitation treatment by contacting the sonicated product with a streptavidin magnetic bead to obtain a target DNA to which a streptavidin magnetic bead is bound Fragment;

(7) Construction of a library based on a target DNA fragment to which a streptavidin magnetic bead is bound.
The method according to claim 1, wherein said genome to be tested is at least a part of a genome obtained by lysing cells or tissues,

Optionally, the cell is a cell line or a primary cell.
The method according to claim 2, wherein said cells or tissues are previously subjected to formaldehyde cross-linking treatment,

Preferably, the cells or tissues are subjected to a transfer treatment by a mouth pipette.
The method of claim 1 wherein the restriction endonuclease is MboI.
The method of claim 1 wherein said biotin labeling process is performed by:

Digestion treatment products with adenine triphosphate deoxynucleotide, guanine deoxynucleotide triphosphate, thymidine triphosphate deoxynucleotide, biotin-labeled cytosine deoxynucleotide and DNA polymerase large fragment The contact was carried out at 37 ° C for 1.5 hours.
The method according to claim 1, wherein said linking treatment is a T4 linkage, and said decrosslinking treatment is carried out by contacting said ligated product with proteinase K, SDS and sodium chloride,

Optionally, the purification treatment is carried out by contacting the decrosslinking treatment product with pre-cooled anhydrous ethanol, the contacting being carried out at -80 ° C for 15 minutes;

Preferably, the decrosslinking treatment product is contacted with hepatic glycogen and sodium acetate during the purification treatment.
The method according to claim 1, wherein the ultrasonication is performed for 134 s under conditions of a Peak Power of 50, a Duty Factor of 20, and a Cycles/Burst of 200.
The method according to claim 1, wherein the database is built by TruSeq, comprising end-repairing a target DNA fragment bound with streptavidin magnetic beads, and terminally adding adenine triphosphate deoxyriborib nucleus. Glycosidic acid and ligation sequencing linker sequences,

Preferably, after the end repair treatment, before the end plus adenine triphosphate deoxyribonucleotide treatment, further comprising Tween washing the magnetic beads treatment;

Preferably, after the terminal plus adenine triphosphate deoxyribonucleotide treatment, before the ligation of the sequencing linker sequence, the method further comprises a tween washing of the magnetic bead treatment;

Preferably, the processing of the sequencing sequencing linker further comprises a Tween wash of the magnetic bead treatment.
The method according to claim 8, further comprising the step of ligating the sequencing linker processing product for DNA first elution treatment and PCR amplification,

Optionally, further comprising combining the PCR amplification product with the AMPure XP magnetic beads,

Optionally, further comprising subjecting the PCR amplification product incorporating the AMPure XP magnetic beads to a second DNA elution treatment.
A sequencing library obtained by the method of constructing a DNA sequencing library of a genome to be tested according to any one of claims 1 to 9.
A method for determining DNA sequence information of a genome to be tested, characterized in that it comprises:

Constructing a DNA sequencing library of the genome to be tested according to the method of any one of claims 1 to 9;

Sequencing the DNA sequencing library to obtain sequencing results;

Based on the sequencing result, DNA sequence information of the test genome is determined.
A method for determining a three-dimensional spatial structure of a genome to be tested, characterized in that it comprises:

Constructing a DNA sequencing library of the genome to be tested according to the method of any one of claims 1 to 9;

Sequencing the DNA sequencing library to obtain sequencing results;

Based on the sequencing result, the three-dimensional spatial structure information of the genome to be tested is determined.