Abstract
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.
Highlights
Great naturalists of centuries-past have catalogued planetary ecosystems at the macroscopic level, primarily for terrestrial and aquatic environments, where organisms were most accessible[1,2].Microscopic life was subsequently given the same attention, again initially focusing on terrestrial and aquatic systems[3,4]
Atmospheric research first described the physicochemical nature of the atmosphere, thereby generating a comprehensive understanding of inanimate components of the troposphere and stratosphere[8]. The origin of these components of air is typically categorised as either inorganic gases or volatile organic compounds (VOCs), the latter of which serve as proxies for the biological activity of organisms[9,10]
Advances in temporal and taxonomic resolution only became possible with the onset of new technologies involving high volumetric flow rate air samplers coupled with metagenomic data generated by next-generation sequencing platforms that had low biomass requirements[22]
Summary
Great naturalists of centuries-past have catalogued planetary ecosystems at the macroscopic level, primarily for terrestrial and aquatic environments, where organisms were most accessible[1,2]. Microbial inhabitants of the third ecosystem of planetary scale, the atmosphere, proved much more difficult to assess due to technological challenges in regard to accessibility These challenges are largely associated with the low-density gaseous state and resulting ultra-low biomass of air[5,6,7]. Advances in temporal and taxonomic resolution only became possible with the onset of new technologies involving high volumetric flow rate air samplers coupled with metagenomic data generated by next-generation sequencing platforms that had low biomass requirements[22]. This approach, which analyses the accessible spectrum of airborne community DNA, enables assessment of the functional complement of airborne microorganisms. The versatility and robustness of the presented pipeline enable analysis of a wide range of environmental settings, both indoor and outdoor, encompassing a wide scope of climatic settings including tropical, temperate, desert and arctic regions
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