Machine-learning guided discovery of emergent antimicrobial activity from dynamic covalent assemblies（2b-RAD-M）

Species-level oral microbiota profiling was performed using 2bRAD-M sequencing in a Streptococcus mutans–induced rat caries model to evaluate treatment-associated microbial community changes. A total of 18 samples were analyzed, with three groups (n = 6 per group): the A5B5 treatment group (A5B5-1, A5B5-2, A5B5-3, A5B5-4, A5B5-5, A5B5-6), the PBS-treated caries control group (Caries-1, Caries-2, Caries-3, Caries-4, Caries-5, Caries-6), and the chlorhexidine (CHX)-treated positive control group (CHX-1, CHX-2, CHX-3, CHX-4, CHX-5, CHX-6). Library preparation and sequencing were conducted by OE Biotech (Qingdao, China) using a Type IIB restriction enzyme-based 2bRAD-M workflow, including BcgI digestion, adaptor ligation, PCR amplification, barcode indexing, and sequencing on the Illumina NovaSeq PE150 platform. For downstream analysis, quality-controlled 2bRAD tags were mapped to a species-resolved 2bRAD marker database constructed from GTDB and Ensembl microbial genomes; candidate taxa were filtered using a Gscore threshold (>5) to reduce false positives, and species-level relative abundances were computed based on mapped reads and species-specific marker counts according to published 2bRAD-M protocols.