DNA提取对微生物多样性测序分析的影响

Influence of DNA Extraction Method on Microbial Diversity Analysis Through Next Generation Sequencing

运用高通量测序技术分析复杂样品中微生物种群的变化情况,已经成为目前微生物研究领域的热点问题之一。而微生物的样品准备,如DNA提取和16S可变区的扩增等,对于测序完成后的数据分析以及微生物原始群落组成的影响是至关重要的。采用国产试剂盒(天根土壤微生物基因组提取试剂盒)和进口试剂盒(MOBIO土壤微生物基因组提取试剂盒)分别对土壤样品和羊瘤胃食糜样品进行DNA提取。然后选取总DNA起始量为25ng,对16S V3可变区进行PCR扩增和文库构建,最后通过数据分析比较不同试剂盒提取的DNA对微生物多样性变化的影响,包括OTU数目、稀释曲线、微生物数量及物种种类等。研究发现,在相同DNA模板量和PCR条件下,进口试剂盒提取的DNA能够获得更多的微生物种类。

Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the most hot issues in the field of microbial research. In this new technology, the microbial sample preparation, such as DNA extraction and the amplification of 16S variable region, etc. , is crucial for the data analysis, especially for the microbial community composition analysis. The soil and sheep rumen chyme samples were used to extract DNA by domestic Kit （TIANamp soil DNA Kit） and imported kit （PowerSoil DNA isolation kit） respectively. Then the 25ng total DNA was used to amplify the 16S V3 region, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the OTU amount, rarefaction curve, microbial number and species were compared by data analysis. It was found that under the same amount of DNA template and PCR cycle number, the DNA treated by imported kit can obtain more microbial species. But under the same DNA extraction kit and DNA template amount, the proportion of the community composition was not the best with more PCR cycle numbers, although the number of species was much more. This may be due to the excessive expansion of the inferior bacteria, which lead to the microbial composition imbalance.