Advancing eDNA analysis techniques of atmospheric bioaerosol samples

Primary biological aerosols in the Earth atmosphere, including pollen, fungal spores, bacteria and viruses, constitute a substantial fraction of aerosols and are actively dispersed by winds over large distances. The composition of bioaerosols is determined by vegetation composition at the surface and influenced by seasonal shifts, weather patterns, and can be modulated by air pollution levels.  

Aerial microbes, which make up less than 1% of airborne entities, have often been overlooked due to challenges in traditional monitoring methods, such as culturing and microscopy. Metagenomics has addressed this gap, allowing for the exploration of species diversity through DNA extraction and culture-independent analyses. This approach is particularly pertinent for understanding enigmatic aerosols, such as pollen, where accurate DNA extraction is essential for precise metagenomic studies, microbial profiling, and aeroallergen detection. Long-read DNA sequencing technologies, such as PacBio and Oxford Nanopore, have transformed biodiversity studies by providing much more comprehensive and accurate genetic information. These technologies produce reads that can cover entire genes or genomes, making them invaluable for studying complex ecosystems. In the field of bioaerosols, long-read sequencing can help to identify new species, detect genetic diversity, and enhance our understanding of microbial community functions, also simplifying the task of genome assembly.  

Metagenomic studies of the atmospheric bioaerosols face challenges due to low concentration of biological material in the air in comparison with water and soil. Overcoming this roadblock, one has to use high-volume samplers (expensive and difficult as well) and/or a highly sensitive and precise procedure of the sample treatment and sequencing. Here the second challenge is addressed by presenting an eDNA analysis procedure applicable to atmospheric samples with moderate-to-low amount of biological material.