Hi-C high-throughput sequencing library building method suitable for microbial metagenomics and application
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A metagenomics and metagenomics technology, which is applied in the field of Hi-C high-throughput sequencing library construction of microbial metagenomics, can solve the problems of single cell library construction schemes, etc., to improve the effective data rate and expand the scope of application Effect
Pending Publication Date: 2020-11-10
WUHAN FRASERGEN CO LTD
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However, for microbial populations in complex natural environments, ordi
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Embodiment 1
[0051] In this example, soil microorganisms were used as the research object, and metagenomics Hi-C high-throughput sequencing was carried out to build and analyze the library. The specific experimental process is as follows:
[0052] 1. Formaldehyde fixation
[0053] 1) Take 1g of fresh soil / intestinal microorganism / marine or river sediment and other metagenomic samples, add 10mL of 1×PBS and mix well;
[0054] 2) Add 1mL of LB medium and mix well, so that the bacteria have sufficient nutrition to ensure immortality;
[0055] 3) Place the centrifuge tube vertically, let it settle naturally for 30 minutes, let the large sand and gravel settle in the lower layer, and the bacteria will swim in the upper layer solution when they are active, and absorb the upper layer solution into a new centrifuge tube;
[0056] 4) Centrifuge at 500g RT for 5min, draw the upper layer solution into a new centrifuge tube;
[0057] 5) Centrifuge at 12000g RT for 5min, and discard the supernatant; ...
Embodiment 2
[0172]In this example, animal excrement was used as the research object of intestinal microorganisms, and metagenomics Hi-C high-throughput sequencing was carried out to build and analyze the intestinal microbial samples. Cells release chromatin, and then undergo steps such as digestion of chromatin, biotin labeling, end connection, library construction, capture of target fragments, and amplification to achieve Hi-C high-throughput sequencing library construction for intestinal microbial metagenomics.
[0173] Among them, the genome integrity, enzyme digestion effect and connection effect are identified by agarose gel electrophoresis pattern, and the agarose gel electrophoresis pattern is as follows: image 3 As shown in B, the decrosslinked electrophoresis strip of the metagenomic group in the figure shows degradation and tailing. Since the metagenome contains bacteria of various physiological stages, it is normal for the decrosslinked DNA to be slightly degraded, and the enti...
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Abstract
The invention relates to a Hi-C high-throughput sequencing library building method suitable for microbial metagenomics and application. The sequencing library building method comprises the following steps that 1), a metagenome sample is taken to be subjected to microbial and impurity separation, and formaldehyde cross-linking is carried out; 2), endonuclease digestion disruption is carried out oncell chromatin to obtain a material after endonuclease digestion; 3), end filling is carried out on the material after endonuclease digestion; 4), DNA cell nucleus intramolecular connection is carriedout; 5), unconnected end biotin is removed to obtain purified DNA, and the purified DNA is subjected to fragmenting, end repairing and A-tailing, and ligation adaptor; 6), DNA target fragments are sorted; and 7), the target fragments are captured by biotin, and library amplification and sequencing are carried out. According to the Hi-C high-throughput sequencing library building method suitable for microbial metagenomics and the application, microorganisms in a complex environment are enriched through separation of the microorganisms and environmental impurities, metagenome data analysis is not limited to a single species any more, and various microorganisms in the complex environment can be subjected to clustering analysis, so that Hi-C high-throughput sequencing library building of microbial metagenomics is achieved, and the application range of the Hi-C technology is expanded.
Description
【Technical field】 [0001] The present invention relates to the technical field of molecular biology, more specifically, to a Hi-C high-throughput sequencing library construction method suitable for microbial metagenomics. 【Background technique】 [0002] Chromosome Conformation Capture (3C) technology is a technique for studying chromosome-protein interaction and chromosome conformation, which can provide detailed information on the association between distant genetic loci, which can be obtained from formaldehyde captured in the nucleus and can be deduced from the three-dimensional folding of chromosomes. In recent years, with the rapid development of second-generation sequencing technology, Hi-C derived from 3C technology takes the entire nucleus as the research object to study the correlation between gene loci in the entire genome. In Hi-C technology, the entire cell line is used as the research object, using high-throughput sequencing technology, combined with bioinformati...
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