Experimental parameters optimisation

Important parameters for sampling, extraction and sequencing were tested and optimised based on absolute (fluorometer and qPCR) and relative abundance assessment (DNA sequencing). Importantly, it should be noted, that only samples collected at an identical time and location may be compared. Therefore, it is mandatory as an experimental setup to deploy multiple air samplers for each set of the parameter optimisation experiments. This is due to the high volatility of biomass concentration and composition of air, particularly when sampling at different time points throughout day and night. The replicability and robustness of this study was, therefore, enabled through simultaneous deployment of up to 12 air samplers at any given time (n = 12).

Comparison to other types of environmental samples: The ultra-low concentration of airborne biomass was investigated relative to other types of environmental samples. To negate possible differences due to sampling location and/or processing method, soil (1 gram per sample extraction), water (1 mL per sample extraction) and air samples (300 L/min, 2 h sampling duration) were collected within the same proximity (in Singapore) and were subsequently processed with identical protocol. Only DNA yield (ng/unit mass or volume of the samples) was assessed for this experiment.

The amassment parameters are sampling duration and sampling flow rate. Sampling duration experiment: With a fixed air flow rate (300 L/min, n = 3), sampling duration was varied at 15, 30, 60, 120 and 180 min. Further, multiple shorter duration samples were also compared to longer duration samples with matching time segments, i.e. first and second 15 min samples were compared to the matching 30 min sample. Air flow rate experiment: With a fixed duration (2 h), three groups of air samplers (n = 4) were run at the same time with varying flow rate at 100, 200 and 300 L/min. The experiments were assessed based on the impact of sampling duration and airflow variations on DNA quantity and microbial composition.

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.