16S rRNA gene amplicon sequencing of the assembled microbial communities by a high-throughput serial transfer culturing.

In nature, microbial communities consist of multiple species that establish complex interactions, including substrate degradation, metabolite exchange, and competition for resources and habitats. Understanding these interactions is crucial for comprehending the determinants of microbial community structure. To identify the large-scale microbial interaction relationships, this study employed serial cultivation of microbiome to analyze combinations of microorganisms that maintain coexistence in various environments. Samples were collected from soils, river water, lake water, and seawater across Japan, and were subjected to high-throughput serial transfer culturing in M9 synthetic medium containing 0.4% various carbon sources. Microbial genome DNA was extracted using the DNeasy UltraClean 96 Microbial Kit (384) (Qiagen), and the V4 region of the 16S rRNA genes was amplified using universal primers 515f and 806r fused to Illumina adapters. Subsequent library preparations (Nextera, Illumina) and 300 bp paired-end sequencing using MiSeq (Illumina) were performed.