Micro-scale spatial metagenomics: revealing high-resolution spatial biogeography of gut microbiomes

Shotgun metagenomics has become a powerful tool for understanding the structure and functioning of gut microbiomes. However, the lack of spatial resolution remains a key limitation of conventional macro-scale shotgun metagenomics, which prevents detailed mapping of microbial organisation within the intestine. To address this, we developed and validated a novel methodology for reconstructing microbial communities at micron-scale resolution. Our micro-scale spatial metagenomics (MSSM) approach integrates cryosectioning, laser micro-dissection, tissue lysis, and Illumina shotgun sequencing library preparation, alongside bioinformatic workflows for quantitative community reconstruction and micro-scale, strain-resolved spatial modelling. We validated the accuracy of MSSM using fluorescence in situ hybridisation of the most prevalent bacteria, and assessed its discriminative power and replicability. Applying MSSM to chicken intestinal samples, we revealed fine-scale spatial variation in microbial community composition, strain-level segregation, and SNP-level microdiversity.