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首页> 外文期刊>Scientific reports. >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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Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform

机译：粪便采样和DNA隔离套件影响使用Miseq Illumina平台的16S rRNA基因测序后的DNA质量和细菌组合物

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摘要

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.
机译：许多研究将人体肠道微生物组的变化与各种疾病发作相关，主要是16S rRNA基因测序。设置最佳采样和DNA隔离程序对于结果的鲁棒性和重现性至关重要。我们对多种采样和DNA分离试剂盒进行了系统的比较，量化了它们对所有分类水平的细菌GDNA质量和细菌组成估计的影响。十六件志愿者测试了三个采样套件。因此，通过两种DNA分离试剂盒加工所有样品。我们发现，粪便采样和DNA隔离套件的选择对细菌组合物的影响相对于革兰氏性，但分离试剂盒具有比采样试剂盒更强的效果。与较低的分类群水平相比，含有分离和抽样试剂盒影响的细菌的比例较高。 PowerLyzer PowerSoil DNA隔离套件优于QIAAMP DNA粪便迷你试剂盒，主要是由于革兰阳性细菌的裂解，同时保持所有其他评估参数在合理范围内的值。当在革兰氏阳性和革兰氏阴性细菌之间的多种研究或计算比进行比较时，需要考虑所提出的效果。

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