Temporal Considerations

When designing an experiment, one must not only consider the spatial scales at which microorganisms live and interact, but as well the temporal scale at which sampling should occur to capture dynamics of interest. Temporal dynamics remain critical in studies of microbially-mediated ecosystem processes (reviewed in 17). Studies have assessed changes in soil microbial communities on temporal scales ranging from days to decades (REFs). Changes over time such as dry-rewetting cycles, introduction of organic matter, pulses of labile substrates (e.g. root exudation), seasonal dynamics and succession of the plant community may in-turn influence the composition and activity of the soil microbial community (18, other REFs). Therefore, careful consideration should be taken when designing experiments in order to sample microorganisms at the relevant temporal scale.
It is also necessary to consider that amplicon sequencing is only a snapshot of microbial prevalence at a given time, resulting in additional challenges regarding analysis and interpretation. The influence of relic or exogenous DNA on investigations of microbial community composition has recently been explored (15, 19, 20)⁠. Relic DNA is extracellular DNA from nonviable cells that has leaked into the environment, the persistence of which can complicate the interpretation of sequencing data by over- or under-estimating microbial diversity (19, 20)⁠. This is of particular concern when temporal dynamics are key to the scientific question. Relic DNA has been estimated to comprise between 30% and 97% of the amplifiable DNA pool (15, 20)⁠. Carini and colleagues demonstrated that the removal of relic DNA from soil samples using propidium monoazide resulted in greater differences in soil communities across timepoints compared to samples where relic DNA removal was not applied (15)⁠. The effects of relic DNA removal were greater in samples across timepoints than in spatially separated samples that were collected at the same time point, again demonstrating the sensitivity of microbial dynamics on a temporal scale.
As an alternative to relic DNA removal, ribosomal RNA (rRNA) amplicon sequencing via complementary DNA (cDNA) synthesis, can be used to reduce the influence of relic DNA on the analysis of community structure. Compared to DNA or other forms of RNA, the lifetime of rRNA is relatively short in the environment (21, 22)⁠. As a result, the changes on community diversity metrics due to rRNA from nonviable cells or from biofilms may be less consequential. However, dormant cells may still contain considerable amounts of RNA (23)⁠. Although growth rates are correlated with rRNA copy numbers in pure cultures, care should be taken not to equate rRNA copy numbers with microbial activity or metabolic state in natural communities (21)⁠. As a result of temporal dynamics within soil microbial communities and the persistence of relic DNA, caution should be applied when inferring shifts in microbial abundance over time. Suggestions for more robust statistical analyses regarding time-series have been discussed in Coenen et al 2020 and others (24 ⁠other REFs).