A method for simultaneous measurement of soil bacterial abundances and community composition via 16S rRNA gene sequencing
A method for simultaneous measurement of soil bacterial abundances and community composition via 16S rRNA gene sequencing
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DOI：
10.1016/j.soilbio.2016.02.003
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发表时间：
2016-05-01
影响因子：
9.7
通讯作者：
Fierer, Noah
中科院分区：
文献类型：
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作者：
Smets, Wenke;Leff, Jonathan W.;Fierer, Noah
最近的许多研究依赖于基于16 S rRNA的测序方法来分析土壤和其他环境样品中发现的细菌或古菌群落。虽然这种方法对于确定给定样品中发现的不同微生物分类群的相对丰度是有价值的，但它没有提供关于目标微生物的丰度如何在样品之间不同的信息。在这里，我们演示了如何在DNA提取步骤中简单添加内标物，从而定量比较细菌16 S rRNA基因的总丰度如何在样品中变化。从两个方面评估了该方法的可靠性。首先，我们用内标物对两种不同土壤的稀释系列进行了加标，以确定我们是否可以准确地量化细胞丰度的差异。我们测试了两种不同的内标物，添加了来自Aliivibrio fischeri或Thermus thermophilus的DNA，这些细菌分类群不太可能在土壤中发现。土壤中16 S rRNA基因的总丰度由序列数据中回收的内标物的16 S rRNA基因的数目计算。这两种标准使我们能够准确地量化土壤中的总基因丰度，因为总16 S rRNA基因估计值与提取的土壤的不同起始量之间存在强正相关性。然后，我们测试了我们是否可以使用这种方法来量化各种土壤类型中微生物丰度的差异;将使用这种方法测量的估计16 S rRNA基因丰度与使用更标准的方法确定的微生物生物量进行比较：磷脂脂肪酸（PLFA）分析和底物诱导呼吸（SIR）分析。用内标测序方法估计的基因丰度与独立的生物量测量值显着相关，并且实际上与SIR和PLFA估计值的相关性比这两个生物量测量值中的任何一个都更好。总之，这些结果表明，在DNA提取之前向土壤或其他环境样品中加入DNA内标物是比较样品中细菌16 S rRNA基因丰度的有效方法。由于在高通量标记基因测序之前容易向土壤样品中添加DNA内标物，现在可以同时和常规地确定16 S rRNA基因丰度和细菌群落组成。(C)2016爱思唯尔有限公司版权所有。
Many recent studies rely on 16S rRNA-based sequencing approaches to analyze bacterial or archaeal communities found in soil and other environmental samples. While this approach is valuable for determining the relative abundances of different microbial taxa found in a given sample, it does not provide information on how the abundances of targeted microbes differ across samples. Here we demonstrate how the simple addition of an internal standard at the DNA extraction step allows for the quantitative comparison of how the total abundance of bacterial 16S rRNA genes varies across samples. The reliability of this method was assessed in two ways. First, we spiked a dilution series of two different soils with internal standards to ascertain whether we could accurately quantify differences in cell abundances. We tested two different internal standards, adding DNA from Aliivibrio fischeri or Thermus thermophilus, bacterial taxa unlikely to be found in soil. The total abundances of 16S rRNA genes in soil were calculated from the number of 16S rRNA genes of the internal standard recovered in the sequence data. Both standards allowed us to accurately quantify total gene abundances in soil as there was a strong positive correlation between total 16S rRNA gene estimations and the different starting amounts of soil extracted. We then tested whether we could use this approach to quantify differences in microbial abundances across a wide range of soil types; comparing estimated 16S rRNA gene abundances measured using this approach to microbial biomass determined with more standard methods: phospholipid fatty acid (PLFA) analysis and substrate induced respiration (SIR) analysis. The gene abundances estimated with the internal standard sequencing approach were significantly correlated with the independent biomass measurements, and were in fact better correlated to SIR and PLFA estimates than either of these two biomass measurements were correlated with one another. Together, these results demonstrate that adding a DNA internal standard to soil or other environmental samples prior to DNA extraction is an effective method for comparing bacterial 16S rRNA gene abundances across samples. Given the ease of adding DNA internal standards to soil samples prior to high-throughput marker gene sequencing, 16S rRNA gene abundances and bacterial community composition can now be determined simultaneously and routinely. (C) 2016 Elsevier Ltd. All rights reserved.