All together now: Limitations and recommendations for the simultaneous analysis of all eukaryotic soil sequences

Abstract

Next generation sequencing technologies have revolutionized microbial ecology, revealing the extensive diversity of bacteria and archaea in our planet (Bates et al., 2011;Thompson et al., 2017), and providing insights into their ecology (Fierer & Jackson, 2006;Martiny et al., 2006). The popularity of amplicon sequencing, where a section of a universal marker gene is amplified and sequenced, has soared over the past decade. In soil, amplicon sequencing of the 16S rRNA gene has been especially useful, as soil prokaryotes remain largely uncultured but are extremely diverse, and perform key ecosystem functions (Steen et al., 2019).Soil eukaryotes, including protists, worms, arthropods, fungi, plant roots, and others, have received comparatively less attention. This is due to technological difficulties associated with sampling, including the complexity and heterogeneity of the soil matrix (Orgiazzi et al., 2016). Consequently, data on the diversity and distributions of soil meso-and macrofauna are limited (Cameron et al., 2019), largely because the identification of these organisms is body size-specific, labour-intensive, and requires a deep knowledge of organisms' morphologies or specific biochemistry (Orgiazzi et al., 2016). Nevertheless, soil eukaryotes are essential to soil functions, as both consumers and ecosystem engineers (Thakur et al., 2019). Amplicon sequencing has become an increasingly attractive alternative for the identification of soil eukaryotes (Pawlowski et al., 2020).Universal marker genes including the ITS region, as well as the 18S rRNA, mitochondrial 16S rRNA, and COI genes, have been used to assess the global diversity of fungi (

Improved high throughput protocol for targeting eukaryotic symbionts in metazoan and eDNA samples

“…As relatively rare taxa will sometimes be of prime interest in antimetazoan metabarcoding studies, factors potentially creating biases against these should be mitigated as far as possible (Fonseca, 2018 ). Other more general considerations for optimal and replicable diversity characterisation of biodiverse samples should also be borne in mind, for example the importance of PCR reagents and conditions (Alberdi et al, 2018 ; Bista et al, 2018 ), chimera formation and secondary structures at lower annealing temperatures (Fonseca et al, 2012 ), artefactual diversity created by incorporation of PCR and sequencing errors, and intragenomic polymorphism between multicopy genes in a single cell (Jurburg et al, 2021 ).…”

Towards all-inclusive community ecology via DNA metabarcoding

“…). In principle, we can amplify the eDNA extracted from one single environmental sample using multiple primers, and then combine the results to attempt an overall reconstruction of biodiversity (Jurburg, Keil, Singh, & Chase, 2021). For example, we might study soil biodiversity by analyzing markers specific for bacteria, fungi, earthworms, insects, springtails.…”

“…Several studies have adopted this approach (Fig. 1b) as it has clear advantages, including relatively cheap cost, and relatively easy implementation (see Jurburg et al, 2021 for additional discussions on limitations and recommendations). In principle, with 2 / 3 primer pairs (e.g.…”

Community metabarcoding reveals the relative role of environmental filtering and dispersal in metacommunity dynamics of soil microarthropods across a mosaic of montane forests

“…We further filtered these community tables by removing ASVs showing abundances of 2 or fewer reads and also those whose contribution to the total number of reads per taxonomic group and library was lower than 1%. Finally, filtered read-count community tables were converted to presence/absence tables (see Jurburg, Keil, Singh, & Chase, 2021). Negative controls were processed alongside actual samples throughout the filtering workflow.…”