Three sets of air samples collected simultaneously (300 L/min, 2 h, n = 4) were subjected to the following storage regimes; direct processing (fresh), −20 °C storage for 5 days (freezer) and room temperature storage for 5 days (RT) and compared for both DNA quantity and microbial profiles.

Parameters optimised for filter processing and DNA extraction were the use of sonication, detergent and impact of pre-incubation. Sonication experiment: Two sets of air samples collected at the same time (300 L/min, 2 h, n = 3) were subjected to filter washing with the room temperature water-bath sonication step included and excluded. Detergent experiment: Four sets of air samples collected at the same time (300 L/min, 2 h, n = 3) were washed with buffer containing four different concentrations of non-ionic detergent Triton-X 100 (%v/v): No detergent (0%), 0.01, 0.1 and 0.5%. Pre-incubation experiment: Three sets of air samples collected at the same time (300 L/min, 2 h, n = 4) were subjected to three different durations of pre-incubation in 55 °C water bath prior to proceeding with the subsequent lysis step of the DNA extraction. The durations were 1 h, 2 h and overnight (14–16 h). These durations were selected to enable the completion of the entire extraction process (filter washing and DNA extraction) within a standard working day (~8 h).

All the above experiments were assessed based on DNA quantity and microbial profiles of the resulting analysis.

The DNA sequencing result was evaluated for the DNA input amount, reproducibility, robustness and taxonomic classification difference between metagenomics and amplicon. DNA input experiment: From a given extracted DNA sample, four different DNA input amounts for direct metagenomic sequencing were tested: 10 ng, 5 ng, 2 ng and 0.5 ng. The number of PCR cycles during library construction were adjusted based on the DNA amount. The final result was assessed based on the taxonomic composition of the sequencing analysis. Reproducibility between replicates: A set of time series samples was analysed to investigate the similarity of the metagenomic profiles between the replicates. The time-series data contains twelve sets of time points with three replicates each. Each set was collected with 300 L/min flow rate and 2-hour sampling duration, spanning across 24 h.Robustness across a range of climatic settings: Air samples collected from locations with different climates (highly variables T and RH) were analysed regarding the success rate of DNA sequencing library construction due to varying amounts and quality of DNA input. 300 L/min flow rate and 2 h sampling duration were used to collect samples in Germany (temperate), Israel (dessert) and Russia (sub-arctic). Comparison of shotgun metagenomic and amplicon marker gene sequencing: The two sequencing approaches were evaluated using taxonomic assignments from identical sets of extracted air samples. DNA samples were split for shotgun metagenomic, 16S bacterial amplicon and ITS fungal amplicon sequencing. The sequencing and analysis methods for the bacterial and fungal amplicon sequencing are detailed in the following section.

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