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).