snow sample Raw sequence reads
Date of Archiving2018
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Key wordssnow; metagenome
Fresh snow sampling A total of 48 snow sampling sites, 44 fresh snow samples from major cities in China, and three in North America and one in Europe, were collected (across a 13,000-km transect belt) in the Northern Hemisphere (23°05′N-66°05′N). Of these, 22 national-scale sampling sites were taken from China in 2016–2017, and 22 sites were further collected in 2018–2019 to verify the previous results. At the same time, in order to better reveal the occurrence, distribution, and inherent underlying mechanism, we also collected four samples from Munich, Washington, New Jersey, and Edmonton, where the level of local air pollution is lower than that of China. These sample sites have different energy structures, population, socio-economic development levels, and air pollution levels. For each location, five replicate samples were taken at each sampling site (100 × 100 m; all four corners and the central point) and measured separately. Details of sampling sites are presented in the Supplementary Table S1. Sampling dates were determined from forecasts for snowfall. Fresh snow samples were taken from the beginning until the end of one snow event. When the snow began to land, it was collected on the ground on top of a plastic sheet; the collected samples were stored in a portable freezer and immediately sent to the laboratory. In the laboratory, the snow samples were melted and mixed at 4 °C, and divided into two portions for DNA extraction and chemical analyses. To ensure a sufficient sample size, from each point, we collected at least 30 L of snow (about 4–8 L snow can melt into 1 L of snow water). The extracted DNA was stored at −80 °C until analysis. Air quality index (AQI) The AQI is used by Chinese government agency to communicate to the public how polluted the air currently is or how polluted it will, most likely, become. There are six pollutant monitoring items in the AQI: sulfur dioxide (SO2), nitrogen dioxide (NO2), inhalable particles (PM10), PM2.5, carbon monoxide (CO2), and ozone (O3). Public health risks increase with increasing AQI levels, and different countries have their own air quality indices, corresponding to different national air quality standards. The computation of the AQI requires an air pollutant concentration over a specified averaging period; it can be calculated either per hour or per 24 h. An individual score (Individual Air Quality Index) is assigned to each pollutant, and the final AQI is the highest of these six scores (for detailed information, see the Supplementary Table S2). Backward trajectory analysis The 2-day (48 h) backward trajectories were calculated every hour at a height of 100 m above the ground level, ending at each snow sampling day, using the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT, NOAA) 4.9 model . The trajectories were then grouped into five clusters using the algorithm of cluster analysis. The clustering of trajectories is based on the total spatial variance method , which minimizes the inter-cluster differences among trajectories while maximizing the outer-cluster differences; it has been widely used in previous studies [22, 28]. Sample chemical analysis Concentrations of NH4+, NO3−, and NO2− were measured using continuous flow analyzers (Germany, SEAL, AA3), while Cl− and SO42− were determined using ion chromatography (USA, Diana, ICS-1000). The Na+, K+, Ca2+, and Mg2+ cations were measured using a full-spectrum direct-reading plasma emission spectrometer (USA, Leeman, Prodigy). Specific-surface area and concentration of particulate matter were measured by a Laser Particle Size Meter (Supplementary Table S3). Socio-economic parameters were collected and calculated from governmental statistical yearbooks, bulletins, and reports (Supplementary Table S1). DNA extraction and HT-qPCR The water from the melted snow (0.5–2 L, depending on the pollution level) was filtered through a 0.22-μm-pore-size filter (diameter, 45 mm; Millipore, New Bedford, MA), which was then used for DNA extraction, using the FastDNA Spin Kit for Soil (MP Biomedicals) according to the manufacturer’s protocol. The DNA quality analysis was performed using a NanoDrop2000 UV-visible spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and 1% (w/v) agarose gel electrophoresis, and the OD260/OD280 ratio of the DNA was between 1.8 and 2.0. The extracted DNA was quantified using the QuantiFluor dsDNA kit (Promega) in a 96-well microplate reader (SpectraMax M5, Molecular Devices), diluted to 50 ng μL−1 using sterile water, and stored at −80 °C for further analysis. A total of 296 primer sets were selected to investigate genes present in the atmospheric snow DNA. These primer sets targeted resistance genes for all major classes of antibiotics (285 primer sets), transposase genes (8 primer sets), one universal class I integron-integrase gene (intI), and one clinical class 1 integron-integrase gene (cintI), and the 16S rRNA gene. The HT-qPCR was performed using the Wafergen Smart Chip Real-time PCR system at the Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences. For each primer set, a non-template negative control was included. The PCR cycle consisted of 10 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing at 60 °C for 30 s. Melting curve analyses were automatically generated by the Wafergen software. All quantitative PCRs were carried out in technical triplicates. Wells with efficiencies beyond the range of 1.7–2.3 or an R2 under 0.99 were discarded. Only data for samples with at least three repeated sampling replicates, which generated amplification products, were regarded as positive and used for further data analysis. The relative copy numbers of ARGs generated by the HT-qPCR were transformed into absolute copy numbers by normalization, using the absolute 16S rRNA gene copy number. The standard curve method of quantification by the Roche 480 system was used to determine the absolute 16S rRNA copy numbers at the same Key Lab facility. Each qPCR reaction mixture (20 μL) contained 10 μL 2× Light Cycle 480 SYBR Green I Master (Roche Applied Sciences), 1 μg μL–1 bovine serum albumin, 1 μM of each primer, 1 ng μL–1 template DNA, and 6 μL nuclease-free PCR-grade water. The thermal cycle consisted of a 10 min of initial enzyme activation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 15 s. A plasmid control containing a cloned and sequenced 16S rRNA gene fragment (1.39 × 1010 copies per liter) was used to generate eight-point calibration curves from tenfold dilutions for standard calculation. All qPCRs were performed in technical triplicates with negative controls. Bacterial 16S rRNA gene sequencing The V4–V5 region of the 16S rRNA gene was amplified, purified, quantified, pooled-, and multiplex-sequenced on an Illumina Miseq platform at Novogene to characterize bacterial communities. Each of the 50-μL PCR reaction mixtures contained 25 μL TaKaRa ExTaq, 0.5 μL bovine serum albumin, 1 μL of each primer, 1 μL DNA as template, and 21.5 μL nuclease-free PCR-grade water. The thermal cycle consisted of 3 min of initial enzyme activation at 94 °C, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min, with a final extension step at 72 °C for 5 min. Raw, paired-end reads were merged to clean sequences after filtering the adapter sequences and removing low-quality reads, ambiguous nucleotides, and barcodes. Raw sequences were demultiplexed and quality-filtered using QIIME  and Mothur .