Monitoring airborne pathogens by nanopore sequencing

Next generation sequencing technologies have revolutionized the field of environmental science. Widely used short-read sequencing enables accurate microbial identification but is often slow, requires large centralised equipment, and does not allow to distinguish highly homologous genomic regions, making the taxonomic classification and genome assembly of closely-related species complicated. Those challenges may be resolved by the implementation of real-time long-read sequencing technologies such as Nanopore sequencing. Nanopore sequencing is a rapid technology that can be employed in situ through portable devices. The long sequencing reads can further improve precision of species identification in mixed microbial communities, and can provide more detailed characterisations of individual microorganisms. Despite its promising application to other environmental samples, nanopore sequencing has not yet been implemented to study bioaerosols and detect pathogens in air samples.Here, we used nanopore sequencing to analyse the environmental DNA extracted from air samples collected in Barcelona, Spain, as an example of a highly urbanised area. As the total amount of DNA found in the air is significantly lower compared to other typical environmental material such as soil or water, we first optimised DNA extraction in combination with the newest nanopore rapid sequencing protocols to achieve a highly accurate genome assembly-based description of the air microbiome. We identified the presence of potentially pathogenic organisms, and annotated the genome assemblies with respect to phenotypic read-outs such as increased virulence and antimicrobial resistance.  We hereby compared the air microbiome assessed through a variety of air sampling methods, including high-volume air samplers, liquid impingers, and standard air filtering approaches. We further assessed if our optimized DNA extraction methods introduced a bias into the described microbiome composition by including positive controls. We were able to demonstrate that it is possible to identify airborne pathogens even when the amount of DNA is low, by leveraging cutting-edge nanopore sequencing technology without requiring cultivation or amplification. This method has the potential to enhance and speed up the surveillance of airborne diseases such as pneumonia, measles, and COVID-19. In upcoming research, we plan to utilise this framework to study the microorganisms present in the air in different settings in order to detect the potential emergence of antimicrobial resistance or highly virulent pathogens in real-time.