Effects of operational taxonomic unit inference methods on soil microeukaryote community analysis using long‐read metabarcoding

Abstract

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large‐scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus's time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic‐dry part that has a species rich grass‐dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%–40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis

“…Another problem linked to the current situation in fungal taxonomy, and therefore in metabarcoding, is the multigene nature of the rRNA markers and the intragenomic variability of the repeats ( 32 – 35 ). All of these factors can unbalance the relative amounts of amplicons, leading to a number of sequences not proportional to the cell densities ( 36 , 37 ), are somehow intrinsic to the biochemical procedures and to the specific nature of multigene markers of rDNA, and are hard to change as long as ITS and LSU are the most important markers in fungal taxonomy ( 6 , 38 ). The settings used aimed at reducing these problems, without pretending to eliminate them, and the results showed that a careful manipulation can produce very high reproducibility among the replicates ( Fig.…”

Considerations for the inclusion of metabarcoding data in the plant protection product risk assessment of the soil microbiome

“…A further consideration for the future of metabarcoding approaches in an environmental risk assessment context is the increasing popularity of long‐read sequencing techniques to study the soil microbiome (Eshghi Sahraei et al, 2022; Tedersoo et al, 2022). The technical specifics of long‐read technologies are beyond the scope of this discussion, but such information can be found in other reviews (Tedersoo et al, 2021).…”

Fine-scale congruence in bacterial community structure from marine sediments sequenced by short-reads on Illumina and long-reads on Nanopore

“…LR environmental metabarcoding has been usually performed on PacBio sequencers, because the Single-Molecule Real-Time (SMRT) technology offers a read quality similar to those of SR platforms. LR metabarcoding is mostly used for taxonomic groups in which SR are too short for a descent assignment, like micro-eukaryotes and specially fungi (Tedersoo et al 2018; Furneaux et al 2021; Kolaříková et al 2021; Eshghi Sahraei et al 2022; Gueidan and Li 2022), but also a few bacterial phyla (Katiraei et al 2022). Despite several published works showed the possibility to use Nanopore sequencing for environmental or food metabarcoding, by sequencing mock communities of known composition (Benítez-Páez et al 2016; Davidov et al 2020; Urban et al 2021; Toxqui Rodríguez et al 2023) or by comparing it with an Illumina library sequenced concurrently (J.…”

“…Third-generation DNA sequencers marked a significant progress for metabarcoding studies, in the fact that the marker size was no longer a technical limitation (tens of kpb for PacBio Sequel II, and no theoretical limit for Nanopore devices), and one can target much more binding sites for primers, improving considerably taxonomic resolution and phylogenetic coverage (Furneaux et al 2021; Tedersoo et al 2021; Eshghi Sahraei et al 2022).…”