DNA of A48
Aerosol samples were collected on 3-micron fluoropore filters at 200 liter per minute using Hi-Q Environmental Products PSU-2 collectors for 24 hours. Samples were then shipped overnight to a molecular laboratory on ice packs and stored in a -80C freezer. To preserve nucleic acid integrity, we transferred the samples into DNA/RNA Shield. To determine optimal conditions for isolation of DNA and RNA, multiple nucleic acid isolation kits, such as MagMAX Viral/Pathogen Ultra, ZymoBIOMICS DNA/RNA Miniprep Kit, and RNeasy PowerFecal Pro kits, were assessed. DNA and RNA concentration was assessed using a Qubit 3.0. RNA was converted into cDNA following evaluation of multiple first- and second-strand cDNA synthesis kits, including SuperScript IV VILO, SuperScript III, and iScript. DNA and converted cDNA were prepared for sequencing using the Oxford Nanopore Technologies (ONT) Rapid PCR Barcoding Kits. PromethION flow cells were used on an ONT P2Solo instrument with up to ten samples per run.SequencingOur preferred methodology for nucleic acid isolation was the ZymoBIOMICS DNA/RNA Miniprep Kit. To sequence RNA samples, SuperScript IV VILO was selected as the method for cDNA conversion due to resistance to inhibitors. Afterwards, we used Invitrogen Second Strand cDNA Synthesis Kit for double-stranded cDNA, then cleaned the resulting product with Invitrogens PureLink PCR Micro Kit. 809 DNA samples and 801 RNA samples were sequenced using ONT Rapid PCR Barcoding Kits.