Optimisation of DNA Extraction from Nasal Lining Fluid to Assess the Nasal Microbiome Using Third-Generation Sequencing.

Sampling nasal lining fluid (NLF) via nasosorption is minimally invasive and well tolerated, but the feasibility of assessing the nasal microbiome using these samples is unknown. However, low biomass makes airway samples particularly susceptible to issues related to contaminant DNA. For this study, we collected nasal swabs and NLF from adult volunteers. DNA was extracted from a mock microbial community and NLF using a column-based kit (ZymoBIOMICS), a precipitation-based kit (Qiagen), or a previously published precipitation-based method. Quality and quantity of DNA was assessed and short-read 16S rRNA sequencing performed to assess feasibility and extraction bias. An optimised methodology was used to extract DNA from NLF and nasal swabs, and long-read 16S rRNA sequencing performed to compare microbial profiles between NLF and nasal swabs. All extraction methods recovered DNA from the mock community, but only precipitation-based methods yielded sufficient DNA from NLF. Extraction methodologies significantly affected microbial profiles, with mechanical lysis needed to minimize bias. Profiles obtained from NLF and swabs were comparable with long-read sequencing. Our findings demonstrate the feasibility of profiling the nasal microbiome using NLF and validated two extraction methodologies as suitable for full-length 16S rRNA sequencing of low-biomass respiratory samples. Our data demonstrate the importance of unbiased DNA extraction methodologies in low-biomass respiratory samples. Additionally, we demonstrated NLF may be an appropriate surrogate for swabs to assess the nasal microbiome.