Experimental and Clinical Research Center, a Cooperation of Charité-Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine, Lindenberger Weg 80, 13125 Berlin, Germany.
Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Charitéplatz 1, 10117 Berlin, Germany.
Cardiovasc Res. 2021 Feb 22;117(3):863-875. doi: 10.1093/cvr/cvaa128.
Recent technical developments have allowed the study of the human microbiome to accelerate at an unprecedented pace. Methodological differences may have considerable impact on the results obtained. Thus, we investigated how different storage, isolation, and DNA extraction methods can influence the characterization of the intestinal microbiome, compared to the impact of true biological signals such as intraindividual variability, nutrition, health, and demographics.
An observative cohort study in 27 healthy subjects was performed. Participants were instructed to collect stool samples twice spaced by a week, using six different methods (naive and Zymo DNA/RNA Shield on dry ice, OMNIgene GUT, RNALater, 95% ethanol, Zymo DNA/RNA Shield at room temperature). DNA extraction from all samples was performed comparatively using QIAamp Power Fecal and ZymoBIOMICS DNA Kits. 16S rRNA sequencing of the gut microbiota as well as qPCRs were performed on the isolated DNA. Metrics included alpha diversity as well as multivariate and univariate comparisons of samples, controlling for covariate patterns computationally. Interindividual differences explained 7.4% of overall microbiome variability, whereas the choice of DNA extraction method explained a further 5.7%. At phylum level, the tested kits differed in their recovery of Gram-positive bacteria, which is reflected in a significantly skewed enterotype distribution.
DNA extraction methods had the highest impact on observed microbiome variability, and were comparable to interindividual differences, thus may spuriously mimic the microbiome signatures of various health and nutrition factors. Conversely, collection methods had a relatively small influence on microbiome composition. The present study provides necessary insight into the technical variables which can lead to divergent results from seemingly similar study designs. We anticipate that these results will contribute to future efforts towards standardization of microbiome quantification procedures in clinical research.
最近的技术发展使得人类微生物组的研究能够以前所未有的速度加速。方法学差异可能对获得的结果产生重大影响。因此，我们研究了不同的储存、分离和 DNA 提取方法如何影响肠道微生物组的特征，以及与个体内变异性、营养、健康和人口统计学等真正的生物学信号相比的影响。
在 27 名健康受试者中进行了一项观察性队列研究。参与者被指示使用六种不同的方法（在干冰上的原始和 Zymo DNA/RNA Shield、OMNIgene GUT、RNALater、95%乙醇、室温下的 Zymo DNA/RNA Shield），每两周收集两次粪便样本。所有样本的 DNA 提取均使用 QIAamp Power Fecal 和 ZymoBIOMICS DNA 试剂盒进行比较。对肠道微生物群进行 16S rRNA 测序，并对分离的 DNA 进行 qPCR。指标包括 alpha 多样性以及对样本的多变量和单变量比较，通过计算控制协变量模式。个体间差异解释了总体微生物组变异性的 7.4%，而 DNA 提取方法的选择则进一步解释了 5.7%。在门水平上，测试的试剂盒在恢复革兰氏阳性菌方面存在差异，这反映在明显偏态的肠型分布中。
DNA 提取方法对观察到的微生物组变异性的影响最大，与个体间差异相当，因此可能会模拟各种健康和营养因素的微生物组特征。相比之下，收集方法对微生物组组成的影响相对较小。本研究为技术变量提供了必要的见解，这些变量可能导致看似相似的研究设计产生不同的结果。我们预计这些结果将有助于为临床研究中微生物组定量程序的标准化做出未来的努力。
