Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based or whole-metagenome sequencing approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective strategy which only sequences ~ 1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles. 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with merely 1 pg of total DNA, high host DNA contamination, or severely fragmented DNA from degraded samples. Tests of 2bRAD-M on various stool, skin, environmental, and clinical FFPE samples suggest a successful reconstruction of comprehensive, high-resolution microbial profiles.

Microbiomes on surfaces in kindergartens, the intermediate transfer medium for microbial exchange, can exert significant impact on the hygiene and wellbeing of young children, both individually and as a community. Here employing 2bRAD-M, a novel species-resolved metagenomics approach for low-biomass microbiomes, we surveyed over 100 samples from seven frequently contacted surfaces by children, plus individual children’s palms, in two kindergartens. Microbiome compositions, although kindergarten-specific, were grouped closely based on the type of surface within each kindergarten. Extensive microbial admixture were found among the various sampled sites, likely facilitated by contact with children’s hands. Notably, bacterial species with potential human health concerns and potentially antibiotic-resistant, although found across all sampled locations, were predominantly enriched on children’s hands instead of on the environmental sites. This first species-resolved kindergarten microbiome survey underscores the importance of good hand hygiene practices in kindergartens and provides insights into better managing hygiene levels and minimizing spread of harmful microbes in susceptible indoor environments.

Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based (e.g., 16S/18S-rRNA) or whole-metagenome sequencing (WMS) approaches. Here, we introduce 2bRAD-M, a "reduction" metagenome-sequencing strategy that simultaneously produces species-level bacterial, archaeal, and fungal profiles for low-biomass samples, yet is cost-competitive with 16S rRNA gene analysis. For mock communities, 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with (i) low biomass of merely 1 pg of total DNA, (ii) high host DNA contamination (99%), and (iii) severely fragmented DNA (50-bp) from degraded samples. Tests of 2bRAD-M on stool, skin and environment-surface samples deliver successful reconstruction of comprehensive, high-resolution microbial profiles with agreement across 16S-rRNA, WMS and existing literature. In addition, it enables microbial profiling in formalin-fixed paraffin-embedded (FFPE) cervical tissue samples which were recalcitrant to conventional approaches due to the low amount and heavy degradation of microbial DNA, and discriminated healthy tissue, pre-invasive cancer and invasive cancer via species-level microbial profiles with 91.1% accuracy. Therefore, 2bRAD-M greatly expands the reach of microbiome sequencing.