Microbiome diversity of low biomass skin sites is captured by metagenomics but not 16S amplicon sequencing.

Established workflows for microbiome analysis work well for high microbial biomass samples, like stool, but often fail to accurately define microbial communities when applied to low microbial biomass samples. Here, we systemically compare microbiome analysis methods -16S rRNA sequencing, shallow metagenomics, and qPCR PMP panels-as well as extraction methods across skin swab samples and mock community dilutions. While extraction method minimally impacted results, with no significant signal for method-specific contamination or bias, we observed critical differences in inferred composition across analysis methods for low biomass samples. Metagenomic sequencing and qPCR revealed concordant, diverse microbial communities on low biomass leg skin samples, whereas 16S amplicon sequencing exhibited extreme bias toward the most abundant taxon. Both qPCR and metagenomics showed that female genital tract bacteria dominated the leg skin microbiome in about half of female subjects. Metagenomics also enabled sub-species analysis, which demonstrated that individuals have consistent within-species diversity across high-biomass forehead and low-biomass leg skin sites. This work illustrates that shallow metagenomics provides the necessary sensitivity and taxonomic resolution to characterize species and strain-level diversity in extremely low biomass samples, opening possibilities for microbiome discovery in previously unexplored niches.