title: 16S amplicon sequencing air control
design: Amplicon sequencing targeting the 16S rRNA, 18S rRNA, as well as the Internal transcribed spacer (ITS) region was used to determine the composition of the bacterial and fungal communities, while qPCR targeting the 16S rRNA and 18S rRNA gene was used to determine the quantity of bacteria and eukaryotes in the samples. DNA was extracted from 9 SBW samples (1 m depth) and 2 river samples, 7 air filters and 3 soil samples (0.25 to 0.28 g), following the DNeasy PowerSoil Pro Kit protocol (HB-2495-002  2018, Qiagen). Two modifications were made to the protocol. In step 2, the PowerBead Pro Tube was vortexed in a TissueLyzer (Qiagen) for 10 min at 40 Hz. In step 16, 50 L of Solution C6 was added to the filter membrane, followed by a 10-min incubation to increase DNA yield. To quantify the amount of bacterial 16S rRNA and eukaryotic 18S rRNA gene copies, a quantitative polymerase chain reaction (qPCR) was performed on a 2 L DNA template. Samples were analyzed using an Mx3005P qPCR instrument (Agilent, Santa Clara, CA, United States). To quantify the 16S rRNA gene copies, universal primers Bac908F (5-AAC TCA AAK GAA TTG ACG GG-3) and Bac1075R (5-CAC GAG CTG ACG ACA RCC-3) were used as described by Lever et al. (2015) 10. Several modifications were applied to the qPCR protocol to improve performance. These included (1) an initial polymerase activation at 95C for 15 min, followed by (2) 40 cycles of PCR. Each cycle consisted of (3) denaturation at 95C for 15 sec, (4) elongation for 15 sec, and (5) fluorescence signal acquisition for 15 sec to monitor amplification in real time. These adjustments aimed to enhance sensitivity and accuracy in target DNA quantification. To quantify the 18S rRNA gene copies, universal primers Euk345F (5-AAGGAAGGCAGCAGGCG-3) and Euk499R (5CACCAGACTTGCCCTCYAAT-3) were used following a method described by Zhu et al. 2005 11. The qPCR was performed with the following cycling conditions: an initial denaturation at 95C for 5 min, followed by 40 cycles of denaturation at 94C for 30 sec, annealing at 58C for 30 sec, and elongation at 72C for 30 sec. A final elongation was carried out at 72C for 10 min. The sandy soil sample from Tyroler river was excluded from the bioinformatic analysis due to a low concentration of gene copies (2.62  10-2 16S rRNA gene copies g1 soil). Libraries were prepared for amplicon sequencing of the V3 and V4 regions of the 16S rRNA gene, the V4 region of the 18S rRNA gene, and the internal transcribed spacer (ITS) for fungal sequencing, all following the Illumina protocol (16S Metagenomic Sequencing Library Preparation, Part # 15044223 Rev. B). The bacterial primers used were Bac341F (5- CCT ACG GGN GGC WGC AG-3) and Bac805R (5-GAC TAC HVG GGT ATCTAA TCC-3), while eukaryotic primers were TAReukFWD1(5-CCAGCASCYGCGGTAATTCC-3) and TAReukREV3 (5-ACTTTCGT TCTTGATYRA-3) 12. The ITS regions (ITS3-KYO2 and ITS4-KYO3) were amplified using forward primer ITS3 (5-GATGAAGAACGYAGYRAA-3) and reverse primer ITS4 (5-CTBTTVCCKCTTCACTCG-3)13. The PCR mixture for both 16S and 18S rRNA reactions contained 25 L template DNA, 2  KAPA HiFi HotStart polymerase (Kapa Biosystems, Inc., Wilmington, MA, United States), 0.5 M forward primer, 0.5 M reverse primer, and BSA (4 g L1). Two L of template DNA were used for seawater and soil samples, and 5 L of template DNA was used for the air samples. The PCR mixture for ITS reactions contained 10 L template DNA, 2  KAPA HiFi HotStart polymerase, 1.0 M forward primer, 1.0 M reverse primer, and BSA (4 g L1). For the first round of PCR, thermal cycling for 16S rRNA included an initial denaturation at 95 oC for 3 min, followed by 20 cycles for seawater and soil extractions and 30 cycles for air sample extractions. Each cycle consisted of denaturation at 95 oC for 30 sec, annealing at 55 oC for 30 sec, elongation at 72 oC for 30 sec, and a final elongation step at 72 oC for 5 min. The thermal cycling conditions for 18S rRNA included an initial step of denaturation at 95 oC for 3 min, followed by two rounds of PCR cycles. For water and soil extractions, 2  10 PCR thermal cycles were performed, while for air sample extractions, 2  15 PCR cycles were used. The conditions for the first round of thermal cycles consisted of denaturation at 95 oC for 30 sec, annealing at 57 oC for 30 sec, and elongation at 72oC for 30 sec. The second round of thermal cycles consisted of denaturation at 95 oC for 3 sec, annealing at 47 oC for 30 sec, and elongation at 72oC for 30 sec, with the final elongation following the protocol for 16S rRNA. Thermal cycling for ITS included an initial denaturation step at 95 oC for 3 min, followed by 30 cycles of denaturation at 95 oC for 30 sec, annealing at 56 oC for 30 sec, elongation at 72 oC for 30 sec, and a final elongation at 72 oC for 5 min. The second round of PCR cycles incorporated the Illumina overhang adapters in the absence of BSA. The second PCR for 16S- and 18S rRNA reactions was run for 10 cycles using the same conditions as for the first PCR for 16S rRNA. For ITS, 8 cycles of PCR were performed using the same conditions as for the first PCR for 16S rRNA.The PCR products were purified using 20 L AMPure XP magnetic beads (Beckman Coulter Inc., CA, USA). Purified PCR products were checked for successful amplification by electrophoresis on a 1.5% agarose gel. The Nextera XT Index primers were incorporated in the third PCR. Each reaction contained 2.5 L of each index primer (N7XX and S5XX), 12.5 L KAPA HiFi HotStart ReadyMix, and 5 L dH2O. The PCR was run for 8 cycles using the same conditions as for the first two PCR, and the PCR product was purified with 20 L AMPure XP beads. The PCR products were quantified using a Quant-iT dsDNA BR Assay Kit on a FLUOstar Omega fluorometric microplate reader (BMG LABTECH, Ortenberg, Germany), then diluted and pooled in equimolar ratios. The pooled library was quantified again using the same assay on a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, United States) which produces two 300-bp long paired-end reads.