Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR.

IF 13.1 1区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Boryana Doyle, Gabriella Z M Reynolds, Mai Dvorak, Dylan G Maghini, Aravind Natarajan, Ami S Bhatt
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引用次数: 0

Abstract

Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.

用16S rRNA qPCR或ddPCR对微生物组中的原核生物进行绝对定量。
原核生物绝对丰度的测量可以为人类肠道微生物组生物学提供重要的见解,并纠正对相对丰度数据的误解。尽管存在几种相对完善的方法来进行这些测量,但大多数微生物组研究并没有报告绝对丰度。为了使具有标准分子生物学能力的研究人员能够将绝对定量纳入他们的微生物组研究,我们提出了一种详细的、逐步的方案,用于严格和可重复的粪便样本中原核浓度的定量。我们包括测量粪便样品含水量的方法,用qPCR或数字液滴PCR (ddPCR)定量16S rRNA原核标记基因的浓度,并分析结果数据。我们还强调并提供了克服定量方法常见缺陷的策略,如16S rRNA基因污染。这种方法的最终输出是16S rRNA拷贝每湿或干克粪便。在样本具有匹配的宏基因组测序信息的情况下,数据可以转换为原核生物的绝对浓度和分类群特异性的绝对浓度。为了使研究人员能够根据他们的具体应用选择合适的方法,我们还比较和对比了qPCR和ddPCR方法。在4天内,通过qPCR或ddPCR从冷冻粪便中提取约80个样品至绝对浓度,无需重新测序。总的来说,该方案提供了一种敏感和直接的方法来测量保存或不保存防腐剂的人类肠道微生物组样品中原核生物的绝对浓度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Nature Protocols
Nature Protocols 生物-生化研究方法
CiteScore
29.10
自引率
0.70%
发文量
128
审稿时长
4 months
期刊介绍: Nature Protocols focuses on publishing protocols used to address significant biological and biomedical science research questions, including methods grounded in physics and chemistry with practical applications to biological problems. The journal caters to a primary audience of research scientists and, as such, exclusively publishes protocols with research applications. Protocols primarily aimed at influencing patient management and treatment decisions are not featured. The specific techniques covered encompass a wide range, including but not limited to: Biochemistry, Cell biology, Cell culture, Chemical modification, Computational biology, Developmental biology, Epigenomics, Genetic analysis, Genetic modification, Genomics, Imaging, Immunology, Isolation, purification, and separation, Lipidomics, Metabolomics, Microbiology, Model organisms, Nanotechnology, Neuroscience, Nucleic-acid-based molecular biology, Pharmacology, Plant biology, Protein analysis, Proteomics, Spectroscopy, Structural biology, Synthetic chemistry, Tissue culture, Toxicology, and Virology.
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