Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform
Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform
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DOI：
10.1038/s41598-019-49520-3
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发表时间：
2019-09-25
影响因子：
4.6
通讯作者：
Budinska, Eva
中科院分区：
文献类型：
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作者：
Videnska, Petra;Smerkova, Kristyna;Budinska, Eva
许多研究将人类肠道微生物组的变化与各种疾病的发病联系起来，主要是通过16S rRNA基因测序。建立最佳的采样和DNA分离程序对结果的稳健性和可重复性至关重要。我们对几种取样和DNA分离试剂盒进行了系统的比较，量化了它们对细菌gDNA质量的影响以及在所有分类水平上的细菌组成估计。16名志愿者测试了三种采样工具。所有样品随后由两个DNA分离试剂盒处理。我们发现，粪便取样和DNA分离试剂盒的选择对细菌组成的革兰氏阳性有影响，但分离试剂盒比取样试剂盒的影响更强。受分离和取样试剂盒影响的细菌比例在较高的分类群水平上大于较低的分类群水平。PowerLyzer PowerSoil DNA分离试剂盒优于QIAamp DNA粪便迷你试剂盒，主要是因为它能更好地裂解革兰氏阳性菌，同时将所有其他评估参数的值保持在合理范围内。在比较多个研究的结果或计算革兰氏阳性和革兰氏阴性细菌之间的比率时，需要考虑到所提出的影响。
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.