High-throughput sequencing database construction technology capable of efficiently capturing three-dimensional conformation of chromosomes of frozen biological sample

A biological sample, high-throughput technology, used in the determination/inspection of microorganisms, biochemical equipment and methods, chemical libraries, etc., can solve the problem of 3D genome research with fewer frozen samples, achieve convenient collection and preservation, and improve library data. The effect of increasing quality and storage capacity

Active Publication Date: 2020-10-02
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, frozen specimens are rarely used for 3D genomic studies

Method used

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  • High-throughput sequencing database construction technology capable of efficiently capturing three-dimensional conformation of chromosomes of frozen biological sample
  • High-throughput sequencing database construction technology capable of efficiently capturing three-dimensional conformation of chromosomes of frozen biological sample
  • High-throughput sequencing database construction technology capable of efficiently capturing three-dimensional conformation of chromosomes of frozen biological sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1, Hi-C high-throughput sequencing of cotton

[0072] In this embodiment, the callus in the cotton tissue culture process was taken as the research object, and two batches of experiments were carried out, wherein the first batch of Hi-C libraries of the two methods was sequenced on a small scale (10g data volume, each Two small data evaluations) to evaluate the quality of the library, and found that the cotton Hi-C library of the new Hi-C method is much higher in the effective data ratio than the traditional method; in the second batch, we built two methods for cotton The libraries (three each) were deeply sequenced, and the genome coverage was approximately 75X, 50X and 30X (FS-Hi-C); 50X, 50X and 30X (traditional Hi-C). It was found that FS-Hi-C significantly increased the effective data ratio of the Hi-C library, and greatly reduced the ratio of repetitive sequences caused by PCR amplification in the library.

[0073] 1. The first batch of tests

[0074] (1...

Embodiment 2

[0216] Example 2, Hi-C high-throughput sequencing of Drosophila

[0217] In this example, Drosophila was used as the research object.

[0218] 1. Sample pretreatment

[0219] The method of the invention :

[0220] 1. Take 5×10 Drosophila cell line (S2 cell line) 6 cells were frozen in liquid nitrogen and ground into a powder.

[0221] 2. Transfer the grinding material to the pre-frozen cross-linking solution at -90°C, and then slowly increase the temperature: -90°C, 6h; -90°C to -60°C, 6h (5°C per hour); -60°C , 6h; -60°C to -30°C, 6h (5°C per hour); -30°C, 6h; -30°C to 0°C, 6h (5°C per hour); each temperature gradient lasted 6h. Replace the water in the cells with the ethanol solution.

[0222] 3. Centrifuge at 3000g at 4°C for 30s, remove the ethanol solution, and then wash three times with NIbuffer buffer placed on ice, after washing, follow the steps of 2×10 6 Add 20 mL of each cell to pre-cooled (pre-cooled on ice) NIbuffer to form a suspension, and shake gently f...

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Abstract

The invention discloses a high-throughput sequencing database construction technology capable of efficiently capturing the three-dimensional conformation of chromosomes of a frozen biological sample.The invention provides a sample pre-processing method suitable for Hi-C high-throughput sequencing library building. The sample pre-processing method includes the steps of freezing a biological sampleto be tested first, then increasing the temperature in a gradient mode, and then cross-linking and fixing. The FS-Hi-C technology is not only suitable for fresh samples, but also for frozen samples.The results show that the three-dimensional structure of chromatin of a frozen replacement sample and a fresh sample are highly similar, and the quality of a Hi-C library is improved. Compared with atraditional Hi-C pretreatment method, FS-Hi-C is highly consistent in chromatin interaction, A / B compartment and TAD and the like, the quality of the library constructed by the FS-Hi-C method is better, the storage capacity is increased, effective interaction data is more, and the repeated data generated by PCR amplification is reduced. The technology reduces the requirements for sample timeliness. The high-throughput sequencing database construction technology provides an improved method for the application of Hi-C in biology.

Description

technical field [0001] The invention relates to the technical field of chromosome conformation capture, in particular to a high-throughput sequencing library construction technology capable of efficiently capturing the three-dimensional chromosome conformation of frozen biological samples. Background technique [0002] Chromosome is a special structure that exists in the nucleus. According to the principle of "structure determines function", the three-dimensional (3D) structure of chromosome naturally becomes the basis for understanding its biological function. It plays a vital role. By exploring the long-range regulation between chromatin, unknown target genes of regulatory elements can be identified, and genes regulated by cis-regulatory elements, phenotypes regulated by eQTL, gene expression regulated by TAD recombination and A / Bcompartment transformation can be studied, but limited by technology For the above reasons, the progress in analyzing the 3D structure of chromo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06C12Q1/6869
CPCC12Q1/6806C40B50/06C12Q1/6869C12Q2523/101C12Q2535/122
Inventor 李付广郑武葛晓阳王晔杨召恩
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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