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Metagenome absolute quantitation method

A genome, unit mass technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of large limitations of the bacterial flora analysis method, save the experimental cost and time, the experimental cost is low, and the operation is simple. easy effect

Active Publication Date: 2020-02-18
康美华大基因技术有限公司
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Problems solved by technology

[0002] In the current 16S and metagenomic sequencing research, the number and structure of the flora are mainly expressed by relative abundance, but the flora analysis method based on the relative value has great limitations

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Embodiment Construction

[0014] The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

[0015] In order to improve the accuracy of the absolute quantitative results of target strains in stool samples, for example, one method is to use flow cytometry to detect the number of bacterial cells in stool samples, and then combine the intestinal flora abundance data obtained by 16S sequencing, Realize the absolute quantification of the bacterial flora; another method is to add a certain amount of external standard sequence to the sample DNA to construct and sequence the 16S amplicon library, and then according to the 16S amplicon readings of the external standard sequence and their absolute A standard curve was drawn for the copy number, and the absolute copy number of the 16S rRNA gene of the species corresponding to the operational taxonomic unit (OTU) representative sequence in the sample was calculated. ...

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Abstract

The invention relates to a metagenome absolute quantitation method. The method comprises the following steps of adding first quantified DNA fragments to samples; extracting genomic DNA on the samples;adding second quantified DNA fragments to the extracted genomic DNA to obtain total genome; performing metagenome library construction and sequencing on the total genome; and through the data of theobtained sequencing results, calculating the bacterial count in the samples in unit mass. The absolute quantitation of bacteria can be obtained by the method, high-cost flow cytometry does not need tobe adopted, and quantified PCR does not need to be performed on specific DNA, so that the process can be simplified and the cost can be reduced.

Description

technical field [0001] The invention relates to a method for absolute quantification of metagenomic groups. Background technique [0002] In the current 16S and metagenomic sequencing studies, the number and structure of the flora are mainly expressed by relative abundance, but the flora analysis method based on the relative value has great limitations. Therefore, achieving absolute quantification of a single strain or target gene is of great significance for studying the function of the flora. In the process of absolute quantification, the difference in DNA extraction yield between different samples (such as feces or soil samples) or even among multiple replicates of the same sample will also significantly affect the absolute quantification results of the target strain. Contents of the invention [0003] The present invention adds two standard DNA fragments (sDNA1 and sDNA2) of known length, sequence and accurate mass respectively before and after the extraction of the g...

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/06
CPCC12Q1/6869C12Q2535/122C12Q2537/16Y02A50/30
Inventor 胡悦许冬瑾蒙洁妙
Owner 康美华大基因技术有限公司
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