Multidomain triple target capture for navigating complex symbioses

Amplicon sequencing remains the benchmark technique for characterizing microbial communities, but the limitations of PCR bias and single-locus targets conspire to limit conclusions. Metagenomics, the primary alternative, shares with amplicon sequencing challenges of economic scaling at adequate sequencing depth. Targeted enrichment strategies can improve the data resolution and economics of sequencing efforts while reducing methodological bias. Here, we develop a target capture strategy for metagenomic characterization of eukaryotic ribosomal DNA and root nodule symbiosis genes and test it in the metagenomes of plant root nodules. We utilize biotinylated RNA probes to selectively capture genomic regions of interest from complex environmental DNA samples, avoiding forms of PCR bias that can undermine community characterization and overcoming the need for conserved priming sites often lacking in functional genes. We designed custom probe sets targeting conserved flanking regions of eukaryotic ITS and known root nodule symbiosis (RNS)-related genes and tested them on diverse root nodule metagenomes and a mock community. We observed high recovery of target loci from samples, very high on-target read proportions, and enhanced detection of low-abundance taxa compared to amplicon sequencing, including stronger alignment with known mock community compositions. This approach will enable deeper insights into the phylogenetic diversity of eukaryotic symbionts, their genomic adaptations, and the functional potential of symbiotic interactions in a cost-effective manner suitable for large-scale projects. This strategy advances our understanding of microbial community dynamics and symbiotic relationships in natural and anthropogenic ecosystems.