Revealing large microbial species diversity hidden behind 16S rRNA OTUs obtained from metabarcoding

Session 3: New approaches to study the gut microbiome ; Communication OS 3

International audience

English. Targeting small parts of the 16S rDNA phylogenetic marker by metabarcoding reveals microorganisms of interest but cannot achieve a taxonomic resolution at the species level, precluding further precise characterizations. To identify species behind OTUs of interest, and particularly those belonging to the rare biosphere, we developed an innovative strategy using gene capture by hybridization that allowed us to recover the corresponding full-length sequences. From three rare OTU sequences of the human gut microbiota which increased upon polyphenol supplementation, we designed specific probes to capture the adjacent sequences of these OTUs from metagenomic DNA libraries. The resulting OTU-enriched libraries were then sequenced and analysed. Using our approach, we revealed 59 nearly full-length 16S rRNA genes sharing ≥ 97% identity with the targeted OTUs and representing 35% of all the retrieved 16S rDNA genes. We highlighted high bacterial diversity hidden behind each OTU and revealed novel taxa. In total, we identified 1 new family belonging to the Clostridiales order, 39 new genera and 52 new species. From each OTU, the revealed 16S rDNA full-length sequences could be highly distant from each other with similarities down to 85%, underlying the poor link between OTU, species and their putative functions. Using a genome mining strategy, we found bacteria related to the novel 16S sequences that are potentially involved in polyphenol degradation. Overall, our results suggest that the human gut microbiota could be much more diverse than previously thought.