Targeted metagenomics using probe capture detect a larger diversity of nitrogen and methane cycling genes in complex microbial communities than traditional metagenomics

Microbes are the key players in the global cycling of nitrogen (N) and carbon (C), controlling the availability and fluxes of C and N in ecosystems, as well as being responsible for losses through the emissions of the powerful greenhouse gasses nitrous oxide (N2O) and methane (CH4). Thus, characterization of microbial functional guilds involved in these processes is high on the scientific agenda. Yet, standard sequence-based characterization methods often reveal only a minor fraction of their diversity in nature due to their frequent low relative abundance, insufficient sequencing depth of traditional metagenomes of complex communities, and limitations in coverage and efficiency of PCR-based assays. Here, we developed and tested a targeted metagenomic approach based on probe capture and hybridization to simultaneously characterize the diversity of multiple key metabolic genes involved in inorganic N and CH4 cycling. We designed comprehensive probe libraries for each of 14 target marker genes, comprising 264,000 unique probes in total. These probes were used to selectively enrich the target genes in shotgun metagenomic libraries. In validation experiments with mock communities of cultured microorganisms, the target gene profiles were similar to those of the original community when sequenced with targeted metagenomics. Furthermore, relative abundances of the marker genes obtained by targeted and shotgun metagenomics from agricultural and wetland soils correlated positively, indicating that the targeted approach did not introduce a significant quantitative bias. However, targeted metagenomics generated substantially higher diversity in terms of taxonomic coverage, and a larger number of sequence reads per sample, which allowed 41 or 1.2 times higher diversity estimates than when using shotgun metagenomics or targeted PCR amplification, respectively. Thus, targeted metagenomics complements current approaches by enabling a targeted, more detailed characterization of the diversity of key functional genes involved in N and CH4 cycling within and between ecosystems. ### Competing Interest Statement The authors have declared no competing interest.