7. Complementary approaches to amplicon sequencing that improve ecological insights

As a consequence of the relative nature of amplicon sequencing data, the majority of such studies are descriptive. Marker-gene base surveys have certainly contributed to generate valuable knowledge regarding microbial diversity and community structure, underpinning the critical roles of microorganisms in the environment. However, the limitation of using DNA sequence information to infer in situ activity, or even potential metabolic functions, has been looming over the field of environmental microbiology from its early days. This inherent property results from both the fact that two organisms with closely-related 16S rRNA gene sequences might possess different metabolic capacities \citep{Li2019},  and even if the function of the organism is known, the presence of DNA or even RNA does not necessarily indicate that the cells are active \cite{Blazewicz2013}. Recent studies are beginning to combine other types of data with amplicon sequencing to improve investigations of ecological patterns. 
Using stable isotopes as an indicator of activity is one of the more popular and robust ways to bridge the gap between microorganisms and their function in ecological processes. In environmental microbiology, DNA or RNA stable isotope probing (SIP) is applied by incubating a sample with a isotopically-labelled substrate (including heavy and rare stable isotopes of C, N, H or O), that can be incorporated into the biomass of metabolically active cells \citep{Angel_2019,Dumont_2005}. Unfortunately, for P no stable isotopes next to the one and only 31P exist. The identity/community profile of the labelled organisms may then be determined using separation of different buoyant densities of the nucleic acids and subsequent sequencing of the different density fractions which allows drawing causal ecological interpretations of the microorganisms active in the uptake and/or assimilation of the substrate. Organisms labelled through SIP may further be detected and identified on a single-cell level using other methods, such as Raman microspectroscopy or NanoSIMS in combination with FISH \citep{Musat_2016,Wang_2016}
Other recent advances in linking microorganisms to functions include so-called 'next-generation physiology' approaches \citep{Hatzenpichler_2020}. Similar to SIP, these methods require the introduction of isotopically labelled or non-canonical molecule into the sample for the detection of metabolically active organisms. The use of heavy-water labelling has become a recent popular approach for universal targeting of all active organisms using either  18O-H2\cite{Aanderud_2011,Schwartz_2007,Angel_2013} or deuterium oxide (D2O) \citep{Li_2019,Eichorst_2015}. The assimilation of  18O-H2O into DNA can be used to deduce microbial growth rates \cite{Hungate2015}, whereas heavy water (D2O) can be detected in the newly synthesized lipids or proteins of active cells  \citep{Li_2019}. Combined with the identification of taxa of interest through amplicon sequencing, next-generation physiology approaches represent powerful tools to bring us to the next step in soil ecological research.
Amplicon sequencing may also be combined with with BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) to target only the fraction of cells within a soil sample that is translationally active in situ \citep{Couradeau_2019,Reichart_2020}. The use of modified indicator molecules opens new avenues for detecting metabolically active cells in the context of environmental samples, however, the application to soil remains limited to very few studies so far \citep{Couradeau_2019,Reichart_2020}. Coupling these labelling approaches to cell sorting via fluorescence-activated cell sorting (FACS) \citep{Couradeau_2019} or Raman-activated cell sorting (RACS) \citep{Lee_2019}, provides a non-destructive alternative to NanoSIMS for identifying the metabolically active organisms, and thus allowing the labelled fraction of cells to be targeted for downstream sequencing. Additionally, combining these labelling approaches with cell sorting and sequencing may further circumvent challenges associated with exogenous DNA.
In addition, amplicon sequencing can certainly also be a valuable tool for planning of more targeted metagenomic or metatranscriptomic studies to investigate phylogenetic composition, functional potential and/or gene expression in the community context \citep{Regalado_2020}.  These approaches remain promising for improving the link between organisms and their ecological roles and circumvent methodological challenges introduced through amplicon sequencing, such as PCR bias. However, both sequencing and bioinformatic costs for gaining functionally relevant insights into ecosystem processes by "omics" approaches are typically orders of magnitudes higher than those needed for analyzing amplicon sequencing data. The use of a limited number of metagenomes or metatranscriptomes in complement to amplicon sequencing presents a cost-effective and informative approach for linking microbial community structure to function in the complex soil environment.