Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.

使用高通量测序进行微生物群落分析部分依赖于 DNA 的保存和 DNA 提取方法的有效性。本研究旨在了解这些参数在多大程度上影响分析。我们获得了用和不用保存溶液处理的样品。此外，我们还比较了 Qiagen 和 Zymo-Research 的 DNA 提取试剂盒。样品的类型是确定的菌株，包括单一物种和混合物，以及来自土壤的未确定的本地微生物群落。我们表明，由于革兰氏阳性细菌的过度代表或革兰氏阴性细菌的代表不足，保存溶液的使用导致了 16S rRNA 基因谱的重大变化。此外，16S rRNA 基因图谱因用于提取的试剂盒类型而异。Zymo 的试剂盒从不同类型的细菌中提取的 DNA 数量大致相等。相比之下，Qiagen 的试剂盒优先从革兰氏阴性菌中提取 DNA，而从革兰氏阳性菌中提取 DNA 的效率较低。试剂盒性能的这些差异极大地影响了我们对微生物生态学研究的解释。