Abstract
BackgroundMicroorganisms comprise the majority of living organisms on our planet. For many years, exploration of the composition of microbial communities has been performed through the PCR-based study of the small subunit rRNA gene due to its high conservation across the domains of life. The application of this method has resulted in the discovery of many unexpected evolutionary lineages. However, amplicon sequencing is subject to numerous biases, with some taxa being missed, and is limited by the read length of second-generation sequencing platforms, which drastically reduces the phylogenetic resolution.ResultsHere, we describe a hybridization capture strategy that allows the enrichment of 16S rRNA genes from metagenomic samples and enables an exhaustive identification and a complete reconstruction of the biomarker. Applying this approach to a microbial mock community and a soil sample, we demonstrated that hybridization capture is able to reveal greater microbial diversity than 16S rDNA amplicon sequencing and shotgun sequencing. The reconstruction of full-length 16S rRNA genes facilitated the improvement of phylogenetic resolution and the discovery of novel prokaryotic taxa.ConclusionsOur results demonstrate that hybridization capture can lead to major breakthroughs in our understanding of microbial diversity, overcoming the limitations of conventional 16S rRNA gene studies. If applied to a broad range of environmental samples, this innovative approach could reveal the undescribed diversity of the still underexplored microbial communities and could provide a better understanding of ecosystem function.
Highlights
Microorganisms comprise the majority of living organisms on our planet
Relative abundances of the different species have been selected so that the final community profile reflects the species abundance variability in an environmental microbial community, with abundant microorganisms (e.g., Clostridium acetobutylicum representing 32.63% of the community) and microorganisms belonging to the rare biosphere (e.g., Methanobrevibacter smithii and Metahnococcus aeolicus, each accounting for 0.00006% of the community)
Here, we demonstrate that hybridization capture targeting the rRNA gene represents a promising approach for microbial community studies
Summary
Exploration of the composition of microbial communities has been performed through the PCR-based study of the small subunit rRNA gene due to its high conservation across the domains of life. The application of this method has resulted in the discovery of many unexpected evolutionary lineages. We report the first application of a hybridization capture strategy [15] that uses a set of probes targeting all known 16S rRNA gene bacterial and archaeal diversity to enrich the full-length biomarker and to explore the microbial diversity of a metagenomic soil sample contaminated with hexachlorocyclohexane (HCH). To evaluate the efficiency of this strategy, we applied this approach to a microbial mock community and we compared it to 16S rRNA gene amplicon sequencing and shotgun sequencing approaches
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