Advancing DNA extraction techniques of atmospheric bioaerosol samples for sequencing with Nanopore without amplification: evaluating purification strategies.. Advancing DNA extraction techniques of atmospheric bioaerosol samples for sequencing with Nanopore without amplification

We present sequencing results of two DNA extraction protocols for atmospheric bioaerosol samples collected on glass-fibre filters widely used in air quality monitoring. The protocols produce high quality molecules suitable for the third-generation sequencing and other applications. The first extraction method includes enzymatic, physical and chemical DNA extraction coupled with SPRI paramagnetic bead technology AMpure XP developed by Beckman Coulter for DNA purification. The second method was the initial protocol from which method one was developed and applied in a Bioaerosol campaign performed in 2021 in Finland and Lithuania. It included a phenol-chloroform step instead of physical disruption for DNA extraction and the paramagnetic beads were not used for DNA purification. The first method was effective for a relatively high mass ratio of biological material and filter: 70 nanograms of potential DNA on the filter to one milligram of the filter fibre, as detected with the second, phenol-chloroform-based, method, but not effective for the ratio lower than 15 nanograms per a milligram of filter. The applicability of new protocol for 3rd generation sequencing was confirmed using Oxford Nanopore technologies (ONT).