Markey Laura, Qu Evan B, Mendall Calen, Finzel Ana, Materna Arne, Lieberman Tami D
Institute for Medical Engineering and Sciences, Massachusetts Institute of Technology; Cambridge, MA, United States.
Department of Civil and Environmental Engineering, Massachusetts Institute of Technology; Cambridge, MA, United States.
bioRxiv. 2025 Jun 24:2025.06.24.661265. doi: 10.1101/2025.06.24.661265.
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.
用于微生物组分析的既定工作流程对高微生物生物量样本（如粪便）效果良好，但应用于低微生物生物量样本时，往往无法准确界定微生物群落。在此，我们系统地比较了微生物组分析方法——16S rRNA测序、浅层宏基因组学和qPCR PMP面板——以及皮肤拭子样本和模拟群落稀释样本的提取方法。虽然提取方法对结果的影响最小，未出现方法特异性污染或偏差的显著信号，但我们观察到，对于低生物量样本，不同分析方法推断出的组成存在关键差异。宏基因组测序和qPCR显示，低生物量腿部皮肤样本上存在一致且多样的微生物群落，而16S扩增子测序对最丰富的分类群表现出极端偏差。qPCR和宏基因组学均表明，约半数女性受试者的腿部皮肤微生物组中，女性生殖道细菌占主导。宏基因组学还实现了亚种分析，结果表明个体在高生物量的额头和低生物量的腿部皮肤部位，种内多样性具有一致性。这项工作表明，浅层宏基因组学为表征极低生物量样本中的物种和菌株水平多样性提供了必要的灵敏度和分类分辨率，为在以前未探索的生态位中发现微生物组开辟了可能性。
