Application of nanopore sequencing for accurate identification of bioaerosol-derived bacterial colonies

Bioaerosol samples are characterized by very low biomass, so culture-based detection remains a reliable and acceptable technique to identify and quantify microbes present in these samples. The process typically involves the generation of bacterial colonies by inoculating the sample on an agar plate, followed by the identification of colonies through DNA sequencing of a PCR-amplified targeted gene. The Sanger method is often the default choice for sequencing, but its application might be limited in identifying multi-species microbial colonies that could potentially form from bacterial aggregates present in bioaerosols. In this work, we compared Sanger and MinION nanopore sequencing techniques in identifying bioaerosol-derived bacterial colonies using 16S rRNA gene analysis. We found that for five out of the seven colonies examined, both techniques indicated the presence of the same bacterial genus. For one of the remaining colonies, a noisy Sanger electropherogram failed to generate a meaningful sequence, but nanopore sequencing identified it to be a mix of two bacterial genera. For the other remaining colony, the Sanger sequencing suggested a single genus with a high sequence alignment and clean electropherogram; however, the nanopore sequencing suggested the presence of a second less abundant genus. These findings were further corroborated using mock colonies, where nanopore sequencing was found to be a superior method in accurately classifying individual bacterial components in mock multispecies colonies. Our results show the advantage of using nanopore sequencing over the Sanger method for culture-based analysis of bioaerosol samples, where direct inoculation to a culture plate could lead to the formation of multispecies colonies.