Highlights
- Soil complexity necessitates careful interpretation of sequencing data
- Studies often do not account for data compositionality, leading to misinterpretation
- Functions should not be inferred from phylogeny as they are rarely conserved
- We discuss complementary approaches that help to improve ecological insights
- We call for journals and authors to improve study reproducibility and data availability
Keywords
amplicon sequencing, soil metabarcoding, soil microorganisms, soil microbial diversity, soil complexity, compositional data
1. Introduction
Soil is one of the most biologically diverse and heterogeneous ecosystems, presenting unique challenges to scientists in the fields of soil and microbial
ecology \citep{Bickel2020}. The critical role of microorganisms as drivers of biogeochemical processes is well-documented, and a major goal of soil ecology remains to decipher the link between the diversity of soil microbial communities, and their function in the environment \citep{Hinsinger_2009,Manzoni_2012}. Historically, studies of microbial communities revealed rather a narrow perspective of the diversity by targeting mainly cultivable bacteria, taxa of high abundance, or microorganisms grouped according to morphological or physiological properties \citep{Staley1985,Steen_2019,Frosteg_rd_2011}. The introduction of next-generation sequencing technologies such as amplicon sequencing has revolutionized our understanding of microbial diversity by enabling the investigation of community composition at a much greater phylogenetic resolution than ever before.
Amplicon sequencing (also termed metabarcoding) is based on PCR-amplification of variable regions of DNA within conserved phylogenetic or functional marker genes \citep{Go_biewski_2019,Semenov_2021} - see also supplementary Table S1 for examples. The accessibility of established assays, the affordability, as well as the availability of free analysis software packages have facilitated the broad use of amplicon sequencing for characterization of the microbiological diversity in environmental samples \cite{Caporaso_2012}. In the field of soil science, its application has accelerated in the last decade as evidenced by the growing number of studies published in specialized soil journals (Fig. 1). The majority of these manuscripts report the analysis of soil community composition and diversity based on phylogenetic marker genes such as the 16S rRNA gene for bacteria and archaea as well as internal transcribed spacer (ITS) regions for fungi. In addition, functional genes can be targeted to obtain information on the organism that may contribute to a specific environmental process \citep{Angel_2018,S_neca_2020,Aigle_2020}.
Such work has enabled researchers to successfully investigate the composition and dynamics of soil microbial communities. Our understanding of microbial diversity has increased dramatically and the activity of microbial communities has now been widely recognized as central in the field of soil science where research questions were historically often tackled from the perspective of individual disciplines such as chemistry, physics, and biology \citep{Baveye_2018}. As evident by the high number of studies being published in recent years, it is safe to say that microbial community analysis via marker-gene sequencing has become a standard tool in soil research. At this stage, it is necessary to discuss potentials, challenges, and pitfalls of the technique applied by soil scientists.