RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Department of Biomedical Engineering, Brno University of Technology, Technicka 12, Brno, Czech Republic.
Sci Rep. 2019 Sep 25;9(1):13837. doi: 10.1038/s41598-019-49520-3.
Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.
许多研究将人类肠道微生物组的变化与各种疾病的发生联系起来，主要通过 16S rRNA 基因测序来实现。建立最佳的采样和 DNA 分离程序对于结果的稳健性和可重复性至关重要。我们对几种采样和 DNA 分离试剂盒进行了系统比较，定量评估了它们对细菌 gDNA 质量和所有分类水平细菌组成估计的影响。16 名志愿者测试了三种采样试剂盒。所有样本随后都通过两种 DNA 分离试剂盒进行处理。我们发现，粪便采样和 DNA 分离试剂盒的选择都会对细菌组成产生影响，特别是对革兰氏阳性菌，但分离试剂盒的影响比采样试剂盒更强。与低分类水平相比，在较高的分类水平上，受分离和采样试剂盒影响的细菌比例更大。PowerLyzer PowerSoil DNA 分离试剂盒的性能优于 QIAamp DNA Stool Mini 试剂盒，主要是因为它能够更好地裂解革兰氏阳性菌，同时保持所有其他评估参数的值在合理范围内。在比较多个研究的结果或计算革兰氏阳性菌和革兰氏阴性菌之间的比值时，需要考虑到这些影响。
