阅读说明：本技术 一种能够高效捕获冷冻生物样本染色体三维构象的高通量测序建库技术 (High-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformation of frozen biological sample ) 是由 李付广 郑武 葛晓阳 王晔 杨召恩 于 2020-06-10 设计创作，主要内容包括：本发明公开了一种能够高效捕获冷冻生物样本染色体三维构象的高通量测序建库技术。本发明提供了适用于Hi-C高通量测序建库的样本前处理方法,包括：将供试生物样本先冷冻,再梯度升温,后交联固定。本发明FS-Hi-C技术不仅适用于新鲜样品,也适用于冷冻样品。本发明结果显示冷冻替换样品和新鲜样品的染色质三维结构高度相似,Hi-C文库质量提高。与传统Hi-C前处理方法相比,FS-Hi-C在染色质互作、A/B compartment和TAD等方面高度一致,且FS-Hi-C法构建文库质量更好,库容增加,有效互作数据更多,PCR扩增产生的重复数据降低。该技术降低了对样本时效性的要求。本发明为Hi-C在生物中的应用提供了一种改进的方法。(The invention discloses a high-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformation of a frozen biological sample. The invention provides a sample pretreatment method suitable for Hi-C high-throughput sequencing library construction, which comprises the following steps: freezing the biological sample to be tested, then heating up in a gradient way, and then fixing by crosslinking. The FS-Hi-C technology is not only suitable for fresh samples, but also suitable for frozen samples. The results of the invention show that the chromatin three-dimensional structures of the frozen replacement sample and the fresh sample are highly similar, and the quality of the Hi-C library is improved. Compared with the traditional Hi-C pretreatment method, the FS-Hi-C method has the advantages that the chromatin interaction, A/B component, TAD and the like are highly consistent, the quality of the constructed library by the FS-Hi-C method is better, the library capacity is increased, effective interaction data are more, and repeated data generated by PCR amplification are reduced. This technique reduces the requirement for sample timeliness. The invention provides an improved method for application of Hi-C in organisms.)

1. A sample pretreatment method suitable for Hi-C high-throughput sequencing and library building comprises the following steps: the biological sample to be tested is frozen and then cross-linked and fixed.

2. The method of claim 1, wherein: the freezing is low-temperature treatment at minus 196 ℃;

further, the freezing is that the biological sample to be tested is put into liquid nitrogen for quick freezing;

and/or

The cross-linking fixation is carried out by using formaldehyde solution;

and/or

And the process of pre-freezing and crosslinking the biological sample to be tested and gradient temperature rise is also included between the freezing and the crosslinking and fixing.

3. The method according to claim 1 or 2, characterized in that: the method comprises the following steps of sequentially processing the biological sample to be tested:

(A1) quick-freezing and grinding by using liquid nitrogen;

(A2) pre-freezing and crosslinking;

(A3) gradient heating;

(A4) centrifuging, adding NIbuffer and filtering;

(A5) formaldehyde crosslinking and fixing;

(A6) glycine terminates crosslinking;

(A7) the cell nuclei were recovered by centrifugation.

4. The method of claim 3, wherein: in the step (a1), the test biological sample is put into liquid nitrogen for quick freezing and ground into powder in the liquid nitrogen;

and/or

The pre-frozen cross-linking of claim 2 is carried out in a pre-frozen cross-linking solution comprising 2% water, 0.01% formaldehyde in an alcohol solution,% by volume;

and/or

In step (A2) of claim 3, transferring the (A1) milled powder to the pre-frozen cross-linking liquid at-90 ℃;

and/or

The gradient temperature rise is 6h at-90 ℃, 6h at-60 ℃, 6h at-30 ℃ and 6h at 0 ℃;

further, in the gradient temperature rise process, the temperature rises from-90 ℃ to-60 ℃ by 5 ℃ per hour, the temperature rises from-60 ℃ to-30 ℃ by 5 ℃ per hour, and the temperature rises from-30 ℃ to 0 ℃ by 5 ℃ per hour;

and/or

In step (a4), the recipe for NIbuffer is as follows: 20mM Hepes pH8, 250mM sucrose,1mM magnesium chloride, 5mM potassium chloride, 40% glycerol by volume, 0.25% Triton X-100 by volume, 0.1mM PMSF, 0.1% beta-mercaptoethanol by volume, 1/5 volume fraction of cocktail;

and/or

In step (a4), removing the supernatant after the centrifugation, adding the NIbuffer suspension precipitate pre-cooled, filtering with Miracloth, collecting the filtrate, and centrifuging;

and/or

The formaldehyde solution of claim 2 which is 37% by volume formaldehyde solution;

and/or

The method of claim 3, wherein in step (A5), the supernatant from the centrifugation in step (A4) is added with 37% by volume of aqueous formaldehyde solution for crosslinking fixation, wherein the crosslinking temperature is room temperature and the crosslinking time is 8 min;

and/or

When the crosslinking and fixing are carried out, the final concentration of the formaldehyde in the system is 1 percent by volume.

5. The method of claim 4, wherein: in step (A6), the crosslinking was terminated by adding a 2.5M glycine solution.

6. A Hi-C high-throughput sequencing and database building method comprises the following steps: pre-treating a test biological sample by the method of any one of claims 1 to 5; the treated samples were then subjected to Hi-C high throughput sequencing for pooling.

7. A Hi-C high-throughput sequencing method comprises the following steps: pre-treating a test biological sample by the method of any one of claims 1 to 5; then carrying out Hi-C high-throughput sequencing on the treated sample to build a library; and finally performing Hi-C high-throughput sequencing.

8. The method according to any one of claims 1-7, wherein: the biological sample is a cell or tissue.

9. The method according to any one of claims 1-8, wherein: the organism is a plant or an animal;

further, the plant is cotton or soybean or radish; the animal is a fruit fly;

further, the biological sample is callus of cotton or a drosophila cell line.

10. Use in any of the following:

(B1) use of the method of any one of claims 1-5 in Hi-C high throughput sequencing pooling;

(B2) use of the method of any one of claims 1-6 in Hi-C high throughput sequencing.

Technical Field

The invention relates to the technical field of chromosome conformation capture, in particular to a high-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformations of a frozen biological sample.

Background

The chromosome is a special structure existing in a cell nucleus, and according to the principle of 'structure determining function', the three-dimensional (3D) structure of the chromosome naturally becomes the basis for understanding the biological function of the chromosome, and the 3D structure of the genome plays an important role in the replication, damage repair and transcriptional regulation of DNA. By exploring the remote control function among chromatins, unknown target genes of the regulatory elements can be identified, and genes regulated by cis-regulatory elements, phenotypes regulated by eQTL, TAD recombination and gene expression regulated by A/Bcomparatent conversion are researched, however, the progress of chromosome 3D structure analysis is slow all the time due to the technical reasons.

In 2002, Dekker et al established 3C chromosome conformation capture (3C) technology, which is a chromosome conformation technology for studying chromosome and protein interaction and can analyze the correlation between gene loci with long linear distance. The 3C technology solves the spatial relationship of 2 DNA fragments with long straight line distance, namely the one-to-one relationship. The technology has milestone significance for the research of the field of chromosome DNA space structure, opens up a new era of the research of chromatin three-dimensional high-grade structure, and inspires a series of derivative technologies. The 4C and 5C technologies appeared in 2006, which solved the relationship of "one-to-many" and "many-to-many" of DNA fragments, respectively. Although these derivation techniques have advanced the understanding of the interrelationships between chromosome fragments, there is still no overall understanding of the chromosome 3D structure.

In recent years, with the development of increasingly sophisticated high throughput sequencing technologies, the acquisition of large-scale genomic information has become easier. In 2009, Dekker et al combined chromosome conformation capture with an increasingly mature high throughput sequencing technology and established the highest throughput Hi-C (highest-throughput chromosome conformation capture) technology for the first time. Hi-C is a high-throughput sequencing technology for analyzing the spatial conformation of chromosomes, can capture the spatial interaction between different gene loci in the whole genome range, and is helpful for researchers to understand the three-dimensional spatial structure of chromosomes, the interaction between chromosomes and the spatial regulation mechanism of gene expression. The Hi-C technology is modified on the basis of the 3C technology, biotin-labeled nucleotides are added, small fragments of DNA generated by subsequent shearing can be enriched, sequencing joints are added to two ends of the fragments, and then the results are compared and analyzed by adopting the latest sequencing means. The technology has the advantages of multiple steps, long consumed time, complex related reagent consumables and more space for improving and optimizing the whole process. These drawbacks of existing Hi-C high throughput sequencing limit the application of this technology to the promotion of functional genomic research, and there is a need for improvements to this technology that provide a new Hi-C high throughput sequencing library-building method that is efficient, convenient, economical and widely applicable. Chromosomal interactions play an important role in genome structure and gene regulation, and Hi-C is a powerful tool for studying the three-dimensional genome structure of species. However, the acquisition of native chromatin conformation requires fresh samples, which hinders the progress of three-dimensional genomic studies.

The solid state of water has three forms, including two crystal forms (hexagonal and cubic) and a glassy state. The glassy state is due to rapid freezing of the sample without time for crystals to form. The formation of crystals causes water to expand when frozen, but the glassy water does not expand after solidification, which makes the glassy water the only ideal frozen form for biological specimens. Based on the above principle, liquid nitrogen (LN2) is widely used for freezing and long-term preservation of biological samples. However, frozen specimens are rarely used for three-dimensional genomic studies. It is generally believed that the three-dimensional genome structure is affected by the sample after being removed from the liquid nitrogen solution during the temperature rise process. The freeze replacement (FS) technique is generally used to maintain the structure of single-and multi-cell animals and plants, and thus it is widely used to prepare samples for conventional optical, transmission and scanning electron microscopes.

Disclosure of Invention

The invention optimizes and improves the key steps of the conventional Hi-C technology which is complicated in sample preparation and not easy to control quality so as to facilitate standardization and quality control, provides more stable and reliable experimental results and promotes further wide application of the Hi-C technology.

In a first aspect, the invention claims a sample pretreatment method suitable for Hi-C high-throughput sequencing and library building.

The sample pretreatment method applicable to Hi-C high-throughput sequencing and library building, which is claimed by the invention, can comprise the following steps: the biological sample to be tested is frozen and then cross-linked and fixed.

Wherein the freezing step can be a low temperature treatment at-196 ℃.

In the invention, the freezing is to put the tested biological sample into liquid nitrogen for quick freezing and grind the sample into powder in the liquid nitrogen.

Wherein, the cross-linking fixation can be cross-linking fixation by using formaldehyde solution. Wherein, the formaldehyde solution can be 37 percent by volume of formaldehyde solution. When the crosslinking and fixing are carried out, the final concentration of formaldehyde in the system is 1 percent by volume.

Further, the process of pre-freezing and crosslinking the biological sample to be tested and gradient temperature rise is also included between the freezing and the crosslinking and fixing. The method can prevent the three-dimensional structural change generated in the temperature rising process after the sample is removed from the liquid nitrogen solution. The biological problem of how to use a low-temperature frozen storage sample for researching the three-dimensional structure of the chromosome is solved, the chromatin conformation in a living cell can be effectively maintained, and the Hi-C data quality of organisms is improved.

Wherein, the pre-freezing crosslinking is carried out in a pre-freezing crosslinking liquid, the pre-freezing crosslinking liquid is an ethanol solution containing 2 percent of water and 0.01 percent of formaldehyde, and the percent represents the volume percentage content.

Wherein the gradient temperature rise is 6h at-90 ℃, 6h at-60 ℃, 6h at-30 ℃ and 6h at 0 ℃. In the gradient temperature rise process, the temperature rises from-90 ℃ to-60 ℃ by 5 ℃ per hour, the temperature rises from-60 ℃ to-30 ℃ by 5 ℃ per hour, and the temperature rises from-30 ℃ to 0 ℃ by 5 ℃ per hour.

Still further, the method may comprise the step of subjecting the test biological sample to the following processes in sequence:

(A1) quick-freezing and grinding by using liquid nitrogen;

(A2) pre-freezing and crosslinking;

(A3) gradient heating;

(A4) centrifuging, adding NIbuffer and filtering;

(A5) formaldehyde crosslinking and fixing;

(A6) glycine terminates crosslinking;

(A7) the cell nuclei were recovered by centrifugation.

In step (a1), the test biological sample was snap frozen in liquid nitrogen and ground to a powder in liquid nitrogen.

In step (A2), the milled powder of (A1) was transferred to a pre-frozen cross-linked solution at-90 ℃ in ethanol containing 2% water, 0.01% formaldehyde,% expressed as volume%.

In step (A3), the gradient temperature rise can be specifically-90 ℃ for 6h, -60 ℃ for 6h, -30 ℃ for 6h, so as to replace the water in the cells with ethanol solution.

In the gradient heating process, the temperature is increased from-90 ℃ to-60 ℃ by 5 ℃ per hour in a gradient manner, and the temperature is increased from-60 ℃ to-30 ℃ by 5 ℃ per hour in a gradient manner.

In step (a4), the recipe for NIbuffer is as follows: 20mM Hepes pH8, 250mM sucrose,1mM magnesium chloride, 5mM potassium chloride, 40% glycerol by volume, 0.25% Triton X-100 by volume, 0.1mM PMSF, 0.1% beta-mercaptoethanol by volume, 1/5 volume fraction of cocktail. Wherein said cocktail is a protease inhibitor. In a specific embodiment of the present invention, the cocktail is a product of MCE, Inc. under the trade designation HY-K0010.

In step (A4), centrifuging (e.g. at 4 deg.C for 30s) to remove supernatant (alcohol solution), adding pre-cooled (ice-cooled) NIbuffer, washing three times as required, and washing 1-2g (e.g. 2g) of the biological sample (or 2 × 10 g)⁶Individual drosophila cell samples) 20mL of the NIbuffer pre-chilled (pre-chilled on ice) was added to form a suspension, then gently shaken for 15min, then Miracloth (Millipore, cat #: 475855) filtering (optionally twice), collecting the filtrate, and centrifuging (e.g. 3000g at 4 deg.C for 15 min).

Further, in the step (a5), an aqueous formaldehyde solution having a concentration of 37% by volume was added to the supernatant obtained by the centrifugation in (a 4). Wherein the final concentration of formaldehyde in the system is 1% volume percentage content. And when the crosslinking and fixing are carried out, the crosslinking temperature is room temperature, and the time is 8 min.

Further, in step (A6), the crosslinking can be terminated by adding a glycine solution (solvent is water) at a concentration of 2.5M.

Further, in step (A7), the centrifugation may be 1500g at 4 ℃ for 5 min. In a second aspect, the invention claims a Hi-C high throughput sequencing and library building method.

The Hi-C high-throughput sequencing and database building method claimed by the invention can comprise the following steps: pre-treating a test biological sample by the method described above; the treated samples were then subjected to Hi-C high throughput sequencing for pooling.

In a third aspect, the invention claims a Hi-C high throughput sequencing method.

The Hi-C high-throughput sequencing method claimed by the invention can comprise the following steps: pre-treating a test biological sample by the method described above; then, performing Hi-C high-throughput sequencing on the processed sample to build a library; and finally performing Hi-C high-throughput sequencing.

In the above three aspects, the biological sample may be a cell or tissue, such as fresh cells or tissue.

In the above three aspects, the organism is a plant (such as cotton, soybean or radish) or an animal (such as fruit fly).

In one embodiment of the invention, the biological sample is specifically callus or Drosophila cell line of cotton.

In a fourth aspect, the invention claims the use of any one of:

(B1) use of a method as hereinbefore described in the first aspect in Hi-C high throughput sequencing pooling;

(B2) use of the method described in the first and second aspects hereinbefore in Hi-C high throughput sequencing.

The invention creatively develops a simple freezing replacement Hi-C technology (Frozen subitute-Hi-C), and the method is not only suitable for fresh samples, but also suitable for Frozen samples stored in liquid nitrogen. The technology applies a freezing replacement (Frozen Substitet) technology to Hi-C sample preparation of a liquid nitrogen (LN2) Frozen sample, and avoids three-dimensional structural change during temperature rising after the sample is removed from a liquid nitrogen solution. The method effectively expands the application range of the Hi-C sample from a fresh sample to a frozen sample, and solves the problems that the sample is difficult to sample and cannot be transported in a long distance. It is noteworthy that the present invention grinds the sample in liquid nitrogen before the freeze replacement (FS) step in plants, which can remove plant cell walls that adversely affect plant cross-linking. The use of cryo-replacement Hi-C replaces the liquid water inside the sample cells with ethanol, so that the frozen sample maintains an unchanged chromatin conformation during warming. The new method is examined in drosophila, cotton, soybean and radish, and the observation of a transmission electron microscope shows that the chromatin structures of a frozen replacement (FS) sample and a fresh sample are highly similar. The chromatin interaction, A/B component and TAD aspects showed a high degree of consistency compared to traditional Hi-C. But the library constructed by the FS-Hi-C method has better quality, increased storage capacity, more effective interaction data and reduced repeated data generated by PCR amplification. The technology reduces the requirement on the timeliness of the sample, and facilitates the collection and storage of the sample. The invention breaks through the limitation that a Hi-C test needs a fresh sample, improves the quality of library data, maintains chromatin conformation, and paves a way for further exploring gene regulation and three-dimensional genome structure.

The technical key point of the invention is that a sample is ground by using liquid nitrogen quick freezing, then pre-crosslinking and gradient heating are carried out, and then crosslinking and fixing are carried out. The invention evaluates the library quality of freezing replacement Hi-C (FS-Hi-C), and finds that in cotton and fruit flies, the three-dimensional conformation of a fresh sample is similar to that of a sample subjected to gradient temperature rise after freezing; and the pretreatment of the sample is carried out by using a freezing replacement mode, so that the data quality of the Hi-C library of cotton and drosophila can be improved. The invention breaks the limitation that a fresh sample must be used in the Hi-C test, and provides wider prospect for the application of Hi-C in biological research.

The invention provides a Hi-C experimental process suitable for organisms such as fruit flies, cotton, soybeans and radishes, solves the technical problems of low cross-linking quality of biological cells and poor establishment of a biological sample library through an innovative and optimized pretreatment method, and obviously improves the effective data volume of the established Hi-C library.

Compared with the prior art, the invention has the following advantages:

(1) in biological experiments, frozen replacement of Hi-C (FS-Hi-C) did not affect the three-dimensional conformation of the chromosome.

(2) In biological experiments, freezing to replace Hi-C (FS-Hi-C) significantly improved the quality of Hi-C library.

(3) In a biological experiment, freezing replacement of Hi-C (FS-Hi-C) effectively expands the application range of a Hi-C sample from a fresh sample to a frozen sample, and solves the problems that the sample is difficult to sample and cannot be transported for a long distance.

(4) In cotton and Drosophila samples, frozen replacement of Hi-C (FS-Hi-C) had no significant effect on chromosome 3D structure.

The FS-Hi-C technology provided by the invention obviously improves the effective data volume of the constructed Hi-C library. Is suitable for the construction of Hi-C libraries of organisms.

Drawings

FIG. 1 is a block diagram of the pretreatment process of the FS-Hi-C high-throughput sequencing and database building method for organisms according to the present invention.

FIG. 2 is a schematic flow chart of the FS-Hi-C method applicable to organisms according to the present invention.

FIG. 3 shows agarose gel electrophoresis measurements of the genomes obtained from two different pre-treatments of the plant samples in the first batch of experiments (small scale sequencing) of example 1. 1 and 8: DNA marker; 2: genome 3 extracted by traditional methods: the genome extracted by the method of the invention; 4: the result of the enzyme digestion (endonuclease DpnII) by the traditional method; 5: the enzyme digestion (endonuclease DpnII) result of the method is obtained; 6: connecting the results after enzyme digestion by the traditional method; 7: the method of the invention links the results after enzyme digestion.

FIG. 4 is a graph of Hi-C matrices constructed at 1Mb resolution using HiCPro software in the first batch of experiments (small scale sequencing) of example 1. In the figure, H and MH are conventional processes and F and CF are the processes of the present invention.

FIG. 5 is a heat map of the cotton chromosome interaction between the cryo-replacement Hi-C (FS-Hi-C) and the conventional Hi-C method treatment in the second batch of experiments (deep sequencing) of example 1.

FIG. 6 is a graph comparing the distribution patterns of frozen replacement Hi-C (FS-Hi-C) and A/B components in cotton treated by the conventional Hi-C method in the second lot of experimental deep sequencing of example 1.

FIG. 7 is a comparison of the topologically related domains (TADs) of cryo-substituted Hi-C (FS-Hi-C) and cotton treated by the traditional Hi-C method in the second trial depth sequencing of example 1.

FIG. 8 is a graph of the change in chromatin structure in nuclei under different treatments in example 1: a. b-f processing modes in the graph; b. chromatin structure of fresh cotton leaves after high pressure freezing and cryo-replacement treatment (HPF-FS-TEM); c. replacing the chromatin structure of the processed karyon by high-pressure freezing and freezing after freezing, grinding and directly heating; d. performing high-pressure freezing and freezing replacement on the chromatin structure of the cell nucleus extracted by the traditional Hi-C method; e. chromatin structure of FS-Hi-C treated cell nucleus extracted without gradient heating process after high pressure freezing and freezing replacement; f. freezing replaces chromatin structure of Hi-C (FS-Hi-C) extracted nuclei after high pressure freezing and freezing replacement.

FIG. 9 is a heat map of the chromosomal interactions of drosophila treated with the frozen replacement Hi-C (FS-Hi-C) and the traditional Hi-C method in example 2.

FIG. 10 is a comparison of distribution patterns of A/Bcomparatents in Drosophila of the frozen replacement Hi-C (FS-Hi-C) and the conventional Hi-C method in example 2.

FIG. 11 is a comparison of topologically related domains (TADs) in Drosophila of example 2 with the frozen replacement of Hi-C (FS-Hi-C) and the traditional Hi-C method.

Detailed Description

The pretreatment flow chart of the FS-Hi-C high-throughput sequencing and database building method suitable for plants is shown in figure 1, and the following treatments are sequentially carried out on a biological sample to be tested: liquid nitrogen quick freezing and grinding, pre-freezing and crosslinking, gradient heating, centrifuging and filtering by NIBbuffer, crosslinking and fixing by 37 percent formaldehyde, and centrifuging and recovering cell nuclei. A schematic flow chart of the FS-Hi-C method applicable to organisms in the invention is shown in FIG. 2.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Pre-freezing the crosslinking liquid: alcohol solution containing 2% of water and 0.01% of formaldehyde,% represents volume percentage.

NIbuffer: 20mM Hepes pH 8; 250mM sucrose; 1mM MgCl₂5mM KCl, 40% (v/v) glycerol, 0.25% (v/v) Triton X-100,0.1mM PMSF, 0.1% (v/v) β -mercaptoethanol, 1/5 volume fraction of cocktail (protease inhibitor, product of MCE, cat. No. HY-K0010).

1.2 × NEBuffer 2: 10 XNEBuffer 2 is a product of NEB company, the product number: B7002S. Diluting as required.

NEB buffer 3.1: NEB buffer3.1 is a product of NEB company, and the product number is: B7203.

10 × NEBuffer 2: NEB company, cat no: B7002S.

1 × NEBuffer 2: 10 XNEBuffer 2 is a product of NEB company, the product number: B7002S. Diluting as required.