Quantitative 16S metagenome sequencing method

A metagenomic, 16SV1-V3 technology, applied in the field of quantitative 16S metagenomic sequencing, can solve problems such as poor comparability, data cannot be directly applied to research topics and testing projects, and cannot be compared, etc., to reduce impact, have horizontal comparability, The effect of improving accuracy

Pending Publication Date: 2020-12-22
ICDC CHINA CDC
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Problems solved by technology

Since the methods adopted by different research topics are not completely consistent, and the deviations introduced by experimental factors and the actual situation are also different, the comparability of the 16S metagenomic sequencing results between different projects is poor. The data of the 16S metagenomic sequencing project published in China cannot be directly applied to its own research topics and testing projects
At the same time, since the 16S metagenomic sequencing data currently only reflect the relative content (ie percentage), the results cannot be directly compared with the results of traditional culture methods, quantitative PCR methods, and digital PCR methods.

Method used

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  • Quantitative 16S metagenome sequencing method
  • Quantitative 16S metagenome sequencing method
  • Quantitative 16S metagenome sequencing method

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Effect test

Embodiment 1

[0061] Example 1 Comparison of Quantitative 16S Metagenome Sequencing Method (Q Method) and Conventional 16S Sequencing Method (N Method)

[0062] 1. Internal reference design

[0063] Two plasmids containing inserted sequences were artificially designed as internal references for sequencing (internal references). The internal reference sequence simulates the 16S V3 / V4 region, and its structure is 341F amplification primer+random sequence (300bp)+805R primer. The inserted random sequence has been verified by the Blast method, and there is no match with known natural species sequences. According to the synthesized plasmid concentration and molecular weight, the number of plasmid copies can be converted. Add 50,000 copies of internal reference 1 and 10,000 copies of internal reference 2 as internal references when building a library for each sample.

[0064] 2. Sampling and sequencing experiments

[0065] Questionnaires were used to investigate the volunteers' personal condi...

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Abstract

The invention discloses a quantitative 16S metagenome sequencing method. Through the adoption of the method of combining random labels with an internal reference method, the accuracy of microbial community structure detection can be effectively enhanced, influences of experiment operation on results can be reduced, and the comparability between sequencing and other molecular biological methods canbe increased; and therefore, the method has important research values for microbial diversity research and unknown pathogen detection.

Description

technical field [0001] The invention relates to the field of sequencing, in particular to a quantitative 16S metagenomic sequencing method. Background technique [0002] 16S rDNA is the most useful and commonly used molecular clock in the taxonomic study of bacteria. It has few types and high content, and exists in all organisms. The 16S rDNA gene sequence includes 9 variable regions and 10 conserved regions. The sequence of the conserved region reflects the relationship between species, while the sequence of the variable region can reflect the difference between species. By designing primers in the two conserved regions of 16S rDNA, the corresponding regions of all bacteria in the sample can be amplified, and because the size of the amplified fragment is moderate, its sequence can be obtained relatively easily by sequencing technology, and the sequencing results can reflect different bacterial genera The difference between them can be compared with 16S databases (such as R...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869G16B30/00G16B40/30
CPCC12Q1/6869C12Q1/689C12Q2600/166G16B30/00G16B40/30C12Q2531/113C12Q2545/101C12Q2535/122
Inventor 张雯韩娜强裕俊彭贤慧张婷婷李秀文
Owner ICDC CHINA CDC
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