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Background  Aerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations.    Results  Here, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures a more comprehensive microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract.    Conclusions  By demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments.

environmental microbiome

Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research