Biogeochemical properties vary across the soil profile and contribute to challenges in interpreting microbial diversity patterns.
Recently, Piwosz et al. \cite{Piwosz2020} enumerated bacterial taxa in aquatic samples and compare these to relative abundances generated by amplicon sequencing. The authors concluded that relative abundance data obtained through amplicon sequencing was robust enough for ecological interpretation on a community level. For specific taxonomic groups, however, the correlation of abundances obtained with both techniques disagreed in large parts, suggesting that care has to be taken when interpreting relative abundance data of single taxa derived from amplicon sequencing. Such technical comparisons for soil samples are rare (e.g. \cite{Ushio_2014}), but given the diversity of soil microbiomes we suggest to use amplicon sequencing data mainly for screenings of soil microbiomes on a community scale (e.g. phylum/class/order level). If the dynamics of certain phylogenetic groups are to be understood on a quantitative basis, we suggest to apply suitable FISH techniques (e.g. \cite{Schmidt_2013}).
As amplicon sequencing is a PCR-based approach, the diversity captured is influenced by the primers selected. More generally speaking, amplicon sequencing inherently does not allow to cover the full diversity of microorganisms and can thus not be used to perform a census of soil organisms. For studies focused on diversity of bacteria and archaea, the 16S rRNA gene is most often amplified...
In mycobiome studies, most primers currently designed and promoted as broadly-fungal specific exclude the deep-branching and evolutionarily ancient fungal or fungal-like organisms in specific phyla (e.g. Chytridiomycota, Glomeromycota and Oomycota). In turn, these taxa must be targeted by group-specific (e.g. Glomeromycota) or broadly eukaryotic primers (e.g. Chytridiocota, Oomycota) to generate amplicons that sufficiently capture such taxonomic groups (Geisen et al. 2019; Lucking et al. 2020; Řezáčová et al. 2019; Stockinger et al. 2010; Wurzbacher et al. 2016). Previous efforts have applied universal fungal primers to analyze arbuscular mycorrhizal fungal communities in both root and soil samples, and reveal similar alpha-diversity estimates using such an approach as compared to sequencing amplicons generated with taxon-specific primers, albeit with much shallower depth. This is because the representation of Glomeromycota in the amplicons generated by supposedly fungal-specific primers is often suppressed as compared to Asco- and Basidiomycota, particularly when working with soil samples (Berruti et al. 2017).
In addition to primer selection challenges, the availability of reference sequences for the different taxonomic groups in databases is variable and not always up-to-date for specific metabarcoding regions. This illustrates challenges with investigating diversity using amplification of a single gene region and suggests the use of phylogenetic analyses together with sequence comparison to databases (e.g. BLAST-based approaches) to correctly assign taxonomic affiliations (Egan et al. 2018; Hart et al. 2015; Stefani et al. XX).