一种新的定量16S rRNA基因扩增子测序方法

A new quantitative 16S rRNA amplicon sequencing method

摘要:

近年来，16S扩增子测序技术被广泛应用于肠道微生物菌群结构和多样性研究，同时也常被用于临床样本中未知病原菌的检测。然而其对样本中物种组成的分辨率只能到属水平的相对丰度，且实验过程中多种因素皆可对结果产生一定影响，如样本起始浓度、PCR循环数、扩增引物等。为解决以上问题，本研究采用随机标签和内参法相结合的方法，开发了一套定量16S扩增子测序方法，将常规的16S rRNA编码基因测序结果中的相对丰度转化为绝对定量的拷贝数，有效提高了肠道菌群结构检测的精准性，降低了实验操作对结果的影响，也提高了测序与其他分子生物学方法间的可比性，有利于未来技术的进一步研发和改进。

关键词:16S rRNA，扩增子，定量，随机标签，内参

Abstract:

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.

Key words:16S rRNA, amplicon, quantitative, random tag, internal marker