Absolute quantification of microbial taxon abundances

Abstract

High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys. The ISME Journal (2017) 11, 584-587; doi:10.1038/ismej.2016 published online 9 September 2016 Recent advancements in high-throughput sequencing of marker genes, such as the 16S rRNA gene, have provided microbial ecologists the tools to accurately infer the relative composition of microbial communities (Franzosa et al., 2015). This resulted in a widespread application of the technology in longitudinal studies where shifts in community structure are related to environmental variables and functional outputs (Faust et al., 2015;Wilhelm et al., 2015). An inherent limitation of the sequencing technology is that the calculated taxon abundances comprise relative values (Widder et al., 2016). Hence, caution must be taken with the biological interpretation of these values, since inter-sample differences in cell density are not considered. To our knowledge, there are no descriptive studies that assess the extent to which relative abundances deliver a skewed image of the actual microbial community dynamics. In this study, we combined robust cell density measurements from flow cytometry (Prest et al., 2013;Van Nevel et al., 2013) with the relative abundances derived from 16S rRNA gene amplicon sequencing. We performed two extensive longitudinal surveys on the central water reservoir of a cooling water system. This engineered freshwater ecosystem was subjected to highly controlled operational phases (Supplementary Information and data set). We quantified the absolute taxon abundances and assessed whether additional insights could be attained with the combined approach.Based on the sample-specified total cell density, the absolute taxon abundances were calculated for each time point. Individual taxon densities ranged from 0.5 to 1 679 cells per μl. Several inter-taxon differences became apparent by performing ordinary least squares regression analysis between the relative and absolute abundances. We focused on the three most abundant taxa, which ...

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