A method for simultaneous measurement of soil bacterial abundances and community composition via 16S rRNA gene sequencing

Abstract

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 25 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 30 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 35 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 40 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 3 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 45 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.

Effects of genetically modified Burkholderia pyrrocinia JK‐SH007E1 on soil microbial community in poplar rhizosphere

“…Increases in enzyme activity may both reflect and stimulate soil microbial activity, thereby increasing the quantities of nutrients available to plants (Monkiedje et al, 2002). As 16S rRNA sequencing estimates only the relative abundance of taxa, the changes in soil bacterial community compositions caused by JK-SH007E1 were difficult to clarify (Smets et al, 2016). Moreover, the beta diversity analysis of soil bacterial communities was not consistent with that of OTU analysis and alpha diversity, probably because the different analysis methods use varied emphasis and judgement criteria (Xue et al, 2008).…”

“…Soil enzyme activities, some of which are simple to measure and show rapid responses to changes in soil microorganism population changes (Monkiedje et al, 2002), have also been widely utilized in the assessment of functional diversity of soil microbial communities (Yao, Min, Lü, & Yuan, 2006). Subsequently, high-throughput sequencing has revolutionized the investigation of soil microbial ecology (Smets et al, 2016 Traditional culture-dependent methods are simple and low cost, but are a poor choice for determining microbial diversity due to large proportions of microorganisms being non-culturable (Rappé & Giovannoni, 2003). In contrast, culture-independent methods give a more complete picture of the actual functions of entire microbial communities .…”

Comparison of Standard Culture-Based Method to Culture-Independent Method for Evaluation of Hygiene Effects on the Hand Microbiome

“…While sequencing is better at showing the entire community, it is not effective in distinguishing living from inactive or dead microbes. In the future, the need for culture plating could be eliminated, if sequencing methods improve to become more quantitative, such as by using a spiking method (39), and lead to resolution of the problem of the inability to distinguish live and/or active microbes from dead and/or dormant bacteria, such as with the propidium monoazide (PMA) method (40) or SYTOX-based methods (41). …”

Soil bacterial quantification approaches coupling with relative abundances reflecting the changes of taxa

“…Smets et al . proposed a method with a simple addition of an internal standard at the DNA extraction step, to simultaneously measure the soil bacterial abundances and community composition via 16 S rRNA gene sequencing 41 . However, there is still a lack of systematic studies combining the absolute and relative measures to reveal the differences of microbial communities in diverse soil ecosystems.…”