Skin microbiome surveys are strongly influenced by experimental design

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provide more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e. gastrointestinal), and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource- and cost-intensive, provides evidence of a community’s functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This work highlights the importance of experimental design for downstream results in skin microbiome surveys. This project folder includes intermediate files generated during the analysis of the dataset and the corresponding analysis scripts used to generate the figures in the paper.

无需培养的皮肤微生物组表征研究愈发常见，这在一定程度上得益于价格亲民且易于获取的测序与分析平台。相较于基于培养的技术，细菌16S核糖体RNA（16S ribosomal RNA, rRNA）基因测序或全宏基因组鸟枪法（whole metagenome shotgun, WMS）测序能够实现更为精准的微生物群落表征。当前主流的实验方案多为表征其他生境（如胃肠道）微生物组而开发，尚未有研究系统比较它们在皮肤微生物组研究中的应用效果。本研究为皮肤研究领域的科研人员构建了一套参考资源，用以指导皮肤微生物组表征相关的实验设计。我们针对16S rRNA基因的两个常用测序区域与WMS测序在还原皮肤微生物组群落组成、多样性以及基因功能富集度方面的表现进行了对比。研究结果显示，WMS测序对微生物群落的还原准确度最高，但16S rRNA基因高变区1-3的测序结果也能提供高度相似的群落信息。而16S rRNA基因高变区4的测序则难以有效捕获皮肤共生微生物，尤其是丙酸杆菌属（Propionibacterium）物种。WMS测序虽具有资源与成本投入较高的特点，能够提供群落功能潜能的相关证据；但基于16S rRNA序列标签的宏基因组预测结果，与WMS测序得到的基因功能谱高度接近。本研究凸显了实验设计对皮肤微生物组研究后续结果的重要影响。本项目文件夹包含该数据集分析过程中生成的中间文件，以及用于绘制论文中各图表的配套分析脚本。