Evaluation of commercial kits and purification approaches for DNA extraction from atmospheric samples for 3rd generation sequencing without amplification.

We present a DNA extraction protocol for atmospheric bioaerosol samples collected on glass-fiber filters widely used in air quality monitoring. The protocol produces high-quality molecules suitable for third-generation sequencing and other applications. The initial protocol was developed and applied in a Bioaerosol campaign performed in Finland and Lithuania in 2021 using low-volume air samplers, which posed stringent requirements to the method sensitivity. The protocol included a phenol-chloroform step for DNA purification, thus involving aggressive reagents; it was also quite time consuming and laborious. The present study advances this protocol to exclude the use of hazardous chemicals by using the SPRI paramagnetic bead technology for DNA purification and compares it to several commercial extraction methods. Despite trailing in efficiency to the initial method, the new development proved to be more efficient than several column-based commercial kits. The updated protocol was effective for a relatively high mass ratio of biological material to filter material: 70 nanograms of potential DNA on the filter to one milligram of filter fiber, as detected with the initial phenol-chloroform-based method. However, the new approach was not effective for a mass ratio lower than 15 nanograms of potential DNA per milligram of the filter material. The applicability of the new protocol for preparation of samples for the 3rd generation sequencing was confirmed by subsequent processing of the samples with the Oxford Nanopore (ONT) GridION sequencer.