Capturing the microbial dark matter in desert soils using culturomics-based metagenomics and high-resolution analysis

Deserts occupy one-third of the Earth's terrestrial surface and represent a potentially significant reservoir of microbial biodiversity, yet the majority of desert microorganisms remain uncharacterized and are seen as "microbial dark matter". Here, we introduce a multi-omics strategy, culturomics-based metagenomics (CBM) that integrates large-scale cultivation, full-length 16S rRNA gene amplicon, and shotgun metagenomic sequencing. The results showed that CBM captured a significant amount of taxonomic and functional diversity missed in direct sequencing by increasing the recovery of amplicon sequence variants (ASVs) and high/medium-quality metagenome-assembled genomes (MAGs). Importantly, CBM allowed the post hoc recovery of microbes of interest (e.g., novel or specific taxa), even those with extremely low abundance in the culture. Furthermore, strain-level analyses based on CBM and direct sequencing revealed that the desert soils harbored a considerable number of novel bacterial candidates (1941, 51.4%), of which 1095 (from CBM) were culturable. However, CBM would not exactly reflect the relative abundance of true microbial composition and functional pathways in the in situ environment, and its use coupled with direct metagenomic sequencing could provide greater insight into desert microbiomes. Overall, this study exemplifies the CBM strategy with high-resolution is an ideal way to deeply explore the untapped novel bacterial resources in desert soils, and substantially expands our knowledge on the microbial dark matter hidden in the vast expanse of deserts.