Stephanie Jurburg, Petr Keil, Brajesh K Singh, Jonathan Chase

The soil environment contains a large, but historically underexplored reservoir of biodiversity. While sequencing of prokaryotic marker genes has become commonplace for the discovery and characterization of soil bacteria and archaea, this approach has been increasingly applied to sequencing eukaryotic marker genes to characterize the diversity of soil eukaryotes. However, understanding the properties and limitations of eukaryotic marker sequences is essential for correctly analyzing, interpreting, and synthesizing the resulting data. Here, we explore how biases in sequencing data arise from variation in morphology, taxonomy and phylogeny, and sampling design, and how these affect measurements of diversity and community composition. We recommend analytical approaches to overcome these limitations, and outline the benchmarking and standardization of sequencing protocols may improve the comparability of the data in the future.