A quantitative sequencing framework for absolute abundance measurements of mucosal and lumenal microbial communities
A quantitative sequencing framework for absolute abundance measurements of mucosal and lumenal microbial communities
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DOI：
10.1038/s41467-020-16224-6
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发表时间：
2020-05-22
影响因子：
16.6
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
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作者：
Barlow, Jacob T.;Bogatyrev, Said R.;Ismagilov, Rustem F.
微生物组研究的一个基本目标是确定哪些微生物影响宿主生理。确定微生物类群变化的标准方法是测量相对丰度，而不是绝对丰度。此外，研究通常只分析粪便，尽管微生物多样性在胃肠道（GI）位置之间存在很大差异。在这里，我们开发了一个定量的框架，以测量绝对丰度的个别细菌类群相结合的精确度与16S rRNA基因扩增子测序的高通量性质的数字PCR。在一项小鼠生酮饮食研究中，我们比较了胃肠道沿着内腔和粘膜样本中的微生物负荷。绝对（而不是相对）丰度的定量测量揭示了生酮饮食中总微生物负荷的减少，并使我们能够确定饮食对粪便和小肠粘膜样品中每个分类群的不同影响。这种严格的定量微生物分析框架适用于不同的胃肠道位置，能够绘制哺乳动物胃肠道的微生物群落图，并更准确地分析微生物组研究中微生物类群的变化。微生物分类群的变化通常来自相对丰度的估计，这固有地限制了分析的深度。在这里，作者提出了一种基于数字PCR锚定和16S rRNA基因测序的绝对定量方法，并展示了生酮饮食如何影响小鼠粪便和小肠粘膜样本中个体分类群的绝对丰度
A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies. Changes in microbial taxa are commonly derived from estimations of relative abundance, which inherently limits the depth of the analysis. Here, the authors present an absolute quantification method based on digital PCR anchoring and 16S rRNA gene sequencing and show how a ketogenic diet affects individual taxon's absolute abundance in stool and small-intestine mucosa samples in mice