title: ITS2 of air sample Amazonia
design: DNA of the particles captured in the air was extracted following Kraaijeveld et al. 2015. The targeted ITS2 region was amplified forward primer ITS3 and reverse primer ITS4 with unique Ion Xpress barcodes for individual sample identification. The PCR reactions (25 l) used: 5 l 5x buffer, 0.5 l 2.5mM dNTPs, 1.25 l 10mM of the respective forward primer and the reverse primer ITS4 coupled with barcodes, 1 l 25mM MgCl2, 0.75 l 100mM BSA, 0.25 l 100% DMSO, 0.5 l Phire HS2 Taq polymerase (Thermo Fisher Scientific), and 4 l of DNA template. PCR program was: 3 min at 98 C, 35 cycles of 5 sec at 98C, 10 sec at 50C, and 30 sec at 72 C, and 5 min at 72 C. An equimolar pool was made by measuring the amplified samples (Qiaxcel) using the DNA Screening Kit (QIAgen) followed by pooling equimolar with the QIAgility (Qiagen) pipetting robot. A clean up of the pool was done with 0,9x (Macherey-Nagel). NucleoMag NGS Clean-up and size select beads and a final quality and quantity check was done with Agilent 2100 Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). The final pool was diluted according to the calculated template dilution factor to target 1030% of all positive ISPs. The template preparation of this pool was carried out on an Ion One Touch instrument with the Ion PGM Template OT2 400 kit (Life Technologies) according to manual 7218 v3.0 and sphere quality control was checked on a Life Qubit 2.0. The ion spheres were loaded on a 318 chip v2 and sequenced with the ion PGM sequencing 400 kit (Life Technologies) on an Ion-Torrent personal genome machine (Life Technologies, ThermoFischer Scientific, USA).