PCR扩增循环数对细菌群落多样性测序分析的影响

运用高通量测序技术分析复杂样品中微生物群落组成及变化趋势,已经成为目前微生物研究领域的热点之一。本研究以复杂土壤样品和应用范围较广的瘤胃食糜样品为对象,选取20、25和30三个扩增循环数分别对样品的16S r RNA基因的V3区进行扩增,然后进行文库构建和测序。最后通过数据分析比较不同的扩增循环数对细菌多样性测定结果的影响。结果表明,扩增循环数越多,捕获到的细菌数量和种类越多;但并非循环数越多,群落中的微生物组成比例最优。整体来看,当扩增循环数为25时,样品中物种的数量和组成是最优的。

Influence of PCR cycle number on microbial diversity analysis through next generation sequencing

Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the hottest issues in the field of microbial diversity research. In this study, the soil and sheep rumen chyme samples were used to extract DNA, respectively. Then the 25 ng total DNA was used to amplify the 16S r RNA V3 region with 20, 25, 30 PCR cycles, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the operational taxonomic unit (OUT) amount, rarefaction curve, microbial number and species were compared through data analysis. It was found that at the same amount of DNA template, the proportion of the community composition was not the best with more numbers of PCR cycle, although the species number was much more. In all, when the PCR cycle number is 25, the number of species and proportion of the community composition were the most optimal both in soil or chyme samples.