Abstract
Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2–5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea).
Highlights
The development of molecular ecology was prompted by indisputable evidence that, for most environments on Earth, the majority of existing organisms had not yet been cultured
The phylogenetic analysis of these environmental sequences (Sanger, .1,200 bp) enabled us to retrieve the affiliations obtained by other authors together with the delineation of freshwater clades corresponding to Cercozoa clade [6] and Perkinsea clades 1 and 2 [7] (Figure S1)
Accuracy of affiliation methods for protist sequences Our taxonomic affiliations were compared with BLAST, a tool commonly used for the identification of microorganisms especially microeukaryotes (e.g., [22]); Ribosomal Database Project Classifier (RDP), which is currently used to classify bacterial and archaeal small sub-unit (SSU) rRNA sequences and fungal LSU rRNA sequences; and STAP implemented in WATERS [41]
Summary
The development of molecular ecology was prompted by indisputable evidence that, for most environments on Earth, the majority of existing organisms had not yet been cultured. This evidence came from the analysis of sequences recovered directly from environmental samples. Vast new lineages of microbial life were uncovered by this approach, changing our picture of the microbial world and yielding a phylogenetic description of community membership [1,2]. Recent studies conducted in lakes have highlighted numerous phylogenetic groups, especially putative parasites (Fungi and Perkinsozoa), and this finding is modifying our view of the microbial loop and the functioning of aquatic ecosystems [6,7]
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