Respiratory tract is home to various microorganisms that impact human health significantly. Regardless, it remains one of the understudied human microbiome sites. Recent studies are migrating towards long-read sequencing platforms that offer improved taxonomic resolution. Samples from the respiratory tract predominantly contain host DNA that can influence sequencing depth and accuracy. Therefore, the suitability of DNA extraction methods should be evaluated. 

We aimed to determine the effects of host depletion methods on oropharyngeal (OP) and nasopharyngeal (NP) swabs from a healthy individual. DNA was extracted with DNeasy Powersoil Pro kit, Saponin/DNase method and NEB Microbiome enrichment kit. DNA copy number in swabs spiked with known concentrations of Staphylococcus aureus and Pseudomonas aeruginosa was evaluated by qPCR. Nanopore MinION sequencing was performed and analysed with Kraken2 and microbiome analyst.  

qPCR data revealed that the host DNA depletion methods successfully depleted >99% host DNA compared to the standard method. A reduction of S. aureus and enrichment of P. aeruginosa was observed with the Saponin/DNase method in contrast to the NEB method. MinION data revealed that samples contain >95-99% host DNA. Interestingly, Saponin/DNase method was successful in depleting ~97% NP host DNA. We observed a microbial dysbiosis index of 0.43 of the OP: NP indicating this individual?s nasopharynx is more enriched with bacteria compared to the oropharynx.  

This highlights the importance of host DNA depletion methods in respiratory microbiome research, but also the importance of the sample type.