Department of Civil Engineering, Kansas State University, Manhattan, KS 66506, USA.
Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011, USA.
J Microbiol Methods. 2023 Mar;206:106677. doi: 10.1016/j.mimet.2023.106677. Epub 2023 Jan 28.
Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.
分析附生叶际微生物群落可能具有挑战性，特别是在应用基于测序的技术时，因为植物衍生的生物分子（如核酸）会产生干扰。最近对附生微生物组的研究综述表明，机械和酶裂解方法都被广泛应用。在这里，我们评估了在不同提取条件下，这两种裂解方法对 DNA 提取产量、纯度、完整性和模板基因组 DNA 每纳克的微生物 16S rRNA 基因拷贝数的影响。此外，还通过 16S rRNA 基因扩增子测序来检测其对细菌群落组成、多样性和重现性的影响。酶裂解方法产生的 DNA 数量比机械裂解方法多一到两个数量级，但 DNA 质量不理想。相反，使用机械方法制备的样品尽管产量较低，但 DNA 纯度较高。出乎意料的是，机械裂解的 DNA 完整性数值（DIN）高于酶裂解。16S rRNA 扩增子测序结果表明，通过机械破碎制备的样品无论提取条件如何，其微生物群落组成都具有可重复性。相比之下，酶裂解方法在不同提取条件下导致分类组成不一致。本研究表明，与酶裂解相比，机械 DNA 断裂更适合附生叶际样品。
