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.

Figure 1

Scatter plot of the absolute and relative abundance of the three most abundant OTUs registered at 79 time points and throughout two time-separated 40-day surveys of a secondary cooling water circuit that operates on a nuclear test reactor. The variance in the relation between absolute and relative abundances increases at elevated values (Breusch–Pagan test, P<0.0001). OTU1 belongs to the betI-A clade. OTU2 and OTU3 belong to the bacI-A clade. Coloured dashed lines depict ordinary least squares regression lines for each OTU. These regressions were used solely for statistical inference and do not necessarily represent the optimal predictive models for these data.

Figure 2

Temporal dynamics for taxa of the two most abundant freshwater clades (that is, bacI-A (OTU2, red; OTU3, orange) and betI-A (OTU1, blue)) during two time-separated 40-day surveys of a secondary cooling water circuit that operates on a nuclear test reactor. The top panel displays the relative abundances (in %) inferred from the 16S rRNA gene amplicon sequencing data. The bottom panel displays the absolute OTU abundances (in cells per μl) and the circle labels represent the total cell density of the microbial community (in cells per μl±s.d.). Horizontal stacked bars highlight different phases of the system during surveillance. Grey zones indicate time periods where the cooling water system was not in operation (control phases), green zones indicate the start-up and blue zones indicate steady-state operation.