Celis Arianna I, Aranda-Díaz Andrés, Culver Rebecca, Xue Katherine, Relman David, Shi Handuo, Huang Kerwyn Casey
Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
iScience. 2022 Feb 11;25(4):103907. doi: 10.1016/j.isci.2022.103907. eCollection 2022 Apr 15.
While microbial communities inhabit a wide variety of complex natural environments, culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.
虽然微生物群落存在于各种各样的复杂自然环境中，但培养能够实现高度可控的条件和高通量检测，从而产生机制性见解。肠道共生菌组合最近被引入作为肠道微生物群的模型，肠道微生物群在宿主健康中发挥着重要作用。然而，尚未建立一种优化财务成本、时间/精力以及准确性/可重复性的16S rRNA测序和样本分析方案。在此，我们系统地确定了具有重大影响、引入偏差和/或可以简化的方案要素。我们的结果表明，群落多样性和组成通常不受大幅简化方案的影响。此外，我们证明，一种严格需氧的嗜盐菌是估计厌氧肠道共生菌群落中绝对丰度的有效内参。这种节省时间和金钱的方案应通过提高16S rRNA数据的可靠性和可比性以及纳入绝对丰度估计来加速发现。
