Study design
As in any study, the sampling or experimental design of microbiome
studies should include sufficient independent replicates, avoiding
confounding effects as much as possible, with the samples representing
appropriate ecological scales given the processes investigated.
Microbiome sampling design must also be well-planned and appropriate to
the specific hypothesis that is being tested. When testing hypotheses
pertaining to the impact of outlier external drivers (e.g., fire,
pollution events, natural disasters), studies would ideally feature
samples that were collected both before and after the event, that are
not confounded by habitat type, geography or physicochemistry. Before
embarking on microbiome studies in the wild, particularly those of which
are opportunistic (i.e., with samples originally collected for other
purposes), researchers should carefully consider if autocorrelation of
factors beyond their control could impede the interpretation of results.
In other words, researchers must be realistic about what can be
accomplished with limited sample sets, since rigorous hypothesis testing
requires equally rigorous sampling protocols and study design.
In addition, the sampling of microbial communities should take into
account their high heterogeneity at small spatial scales due to
micro/mesoscale heterogeneity of their environment (Vos et al., 2013;
Zhang et al., 2014) or neutral assembly dynamics (Woodcock et al.,
2007)). For example, composite samples (i.e. pooled individual samples)
can be combined prior to homogenisation and sub-sampling, in order to
reduce the local, micro-scale heterogeneity if it is irrelevant to the
questions being studied (George et al., 2019). Here, knowledge of how,
and at what scale, the target community responds to external drivers
will inform adequate sampling design. For example, a composite 0.2mg
sediment sample is likely to be representative of the bacterial,
archaeal, and microbial eukaryotic biospheres, but will not sample
microscopic invertebrate taxa effectively, due to issues of scale
(Nascimento et al., 2018). Smaller samples will contain some microscopic
taxa and trace environmental DNA but they are inadequate at representing
the underlying meio- and macro-faunal communities. As the target
organisms grow in size, the sample volume and spatial extent of the
studied area should be correspondingly expanded.