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

Abstract

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 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.