Method for eliminating host DNA contamination for use in viral metagenomic analysis of water environment

A metagenome and host technology, applied in the field of water environment virus metagenome analysis, can solve problems such as affecting sequencing results, and achieve the effect of increasing research process and cost, and ensuring quality

Inactive Publication Date: 2018-07-31
NORTHEAST INST OF GEOGRAPHY & AGRIECOLOGY C A S
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention aims to solve the problem that there are a small amount of host cells in the virus concentrate in the water environment virus metagenome research, which affects the sequencing results, and provides a method for eliminating host DNA pollution for analysis of the water body environment virus metagenomics

Method used

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  • Method for eliminating host DNA contamination for use in viral metagenomic analysis of water environment
  • Method for eliminating host DNA contamination for use in viral metagenomic analysis of water environment
  • Method for eliminating host DNA contamination for use in viral metagenomic analysis of water environment

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specific Embodiment approach 1

[0022] Specific embodiment one: this embodiment is implemented according to the following steps by eliminating host DNA pollution and being used for the method for water body environment virus metagenomic analysis:

[0023] 1. Separation and concentration of water virus particles:

[0024] a. Collect water bodies in the field to obtain water samples;

[0025] b. The water sample is filtered through multi-layer nylon gauze, and then the bacteria are removed through the filter column of the tangential flow filtration system to obtain the virus particle solution;

[0026] c. The virus particle solution is concentrated through the filter column of the tangential flow filtration system to obtain the virus particle concentrate;

[0027] d. Add the virus particle concentrate to the ultracentrifuge tube, add a sucrose solution with a mass concentration of 19% to 21% at the bottom of the ultracentrifuge tube, and then perform centrifugation to obtain the centrifuged virus particle con...

specific Embodiment approach 2

[0038] Embodiment 2: This embodiment is different from Embodiment 1 in that the water sample is filtered through multiple layers of 400-mesh nylon gauze in step b.

specific Embodiment approach 3

[0039] Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that the centrifugation in step d is performed at 4° C. at a speed of 28,000 rpm for 2 hours.

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Abstract

The present invention discloses a method for eliminating host DNA contamination for use in viral metagenomic analysis of water environment, belongs to the field of biotechnology, and aims to solve theproblem of a small amount of host cells in a virus concentrate affect sequencing results in the viral metagenomic research of the water environment. The method for viral metagenomic analysis of the water environment is as follows: 1, filtering a water sample through gauze, removing bacteria by a filter column of a filtration system, concentrating, centrifuging, and preparing a virus particle ultra-speed-centrifuge concentrate; 2, adding DNase and RNase working solutions, performing PCR amplification, detecting whether digestion is complete, if the digestion is not complete, adding lysozyme tolyse bacterial cells, continuing digestion, and inactivating nuclease at high temperature to obtain a virus particle concentrate without host genetic material contamination; and 3, amplifying virus whole genome. The sample is treated by the lysozyme for release of DNA of the contaminated bacterial cells, then the DNA is digested by the nuclease, and the operation is simple, and the applicabilityis strong.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for analyzing the metagenome of water environment viruses. Background technique [0002] Metagenomics uses next-generation high-throughput sequencing (NGS) to analyze microbial diversity, population structure, and evolutionary relationships, and further explore the relationship between microbial population functions, interactions, and the environment. potential biological significance. Viruses are the most numerous living organisms on earth, conservatively estimated at 10 31 above. Compared with other microorganisms with cell form, such as bacteria and fungi, the academic community's understanding of the diversity, genetic composition and ecological functions of viruses in the natural environment is still in its infancy. Viruses are often referred to as the "dark matter" unknown to the earth's organisms . Applying the research method of metagenomics to the st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806
Inventor 王光华孙岩王新珍刘俊杰
Owner NORTHEAST INST OF GEOGRAPHY & AGRIECOLOGY C A S
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