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Abstract
Microbial community analysis via marker gene amplicon sequencing has become a standard application in soil research. ddThe spatio-temporal complexity of soil and the diversity of organisms therein, however, require careful analysis of what information can be extracted from the data to prevent mis- or over-interpretation. In this perspective, we describe the technical challenges and limitations of amplicon sequencing studies in soil and present statistical and experimental approaches that can help addressing the compositional nature of the data. We illustrate the impact of compositionality on the interpretation of relative abundance data and discuss effects of sample replication on the statistical power in soil community analysis. Additionally, we argue for the need for increased study reproducibility and data availability, as well as complementary techniques for generating deeper ecological insights into microbial roles and our understanding thereof in soil ecosystems. At this critical turning point in the field of soil ecology, we call upon researchers and specialized soil journals to consider the current state of data analysis, interpretation and availability to improve the rigor of future studies.
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 (taxonomic) or functional marker genes \citep{Go_biewski_2019,Semenov_2021} - see also supplementary Table S2 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 is becoming a standard tool in soil research. This marks a critical turning point in the field and makes it necessary to discuss potentials, challenges, and pitfalls of the technique applied by soil scientists.