Investigation of the microbial ecology of terrestrial, aquatic and atmospheric ecosystems requires specific sampling and analytical technologies, owing to vastly different biomass densities typically encountered. In particular, the ultra-low biomass nature of air presents an inherent analytical challenge that is confounded by temporal fluctuations in community structure. Our ultra-low biomass pipeline advances the field of bioaerosol research by significantly reducing sampling times from days/weeks/months to minutes/hours, while maintaining the ability to perform species-level identification through direct metagenomic sequencing. The study further addresses all experimental factors contributing to analysis outcome, such as amassment, storage and extraction, as well as factors that impact on nucleic acid analysis. Quantity and quality of nucleic acid extracts from each optimisation step are evaluated using fluorometry, qPCR and sequencing. Both metagenomics and marker gene amplification-based (16S and ITS) sequencing are assessed with regard to their taxonomic resolution and inter-comparability. The pipeline is robust across a wide range of climatic settings, ranging from arctic to desert to tropical environments. Ultimately, the pipeline can be adapted to environmental settings, such as dust and surfaces, which also require ultra-low biomass analytics.

由于通常遇到的生物量密度差异很大，因此对陆地、水生和大气生态系统的微生物生态学进行调查需要特定的采样和分析技术。特别是，空气的超低生物量性质提出了一个固有的分析挑战，该挑战被群落结构的时间波动所混淆。我们的超低生物量管道通过将采样时间从数天/数周/数月显着减少到数分钟/数小时，同时保持通过直接宏基因组测序进行物种水平鉴定的能力，从而推进了生物气溶胶研究领域。该研究进一步解决了所有影响分析结果的实验​​因素，例如积累、储存和提取，以及影响核酸分析的因素。使用荧光测定法、qPCR 和测序评估每个优化步骤的核酸提取物的数量和质量。宏基因组学和基于标记基因扩增（16S 和 ITS）的测序都根据它们的分类分辨率和相互可比性进行了评估。该管道在从北极到沙漠再到热带环境的各种气候环境中都很稳健。最终，管道可以适应环境设置，例如灰尘和表面，这些也需要超低生物量分析。该管道在从北极到沙漠再到热带环境的各种气候环境中都很稳健。最终，管道可以适应环境设置，例如灰尘和表面，这些也需要超低生物量分析。该管道在从北极到沙漠再到热带环境的各种气候环境中都很稳健。最终，管道可以适应环境设置，例如灰尘和表面，这些也需要超低生物量分析。