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Unlocking the Functional Capacity of Sinonasal Microbiota Using Microbial DNA Enrichment Techniques
Monday, March 7, 2016
South Exhibit Hall H (Convention Center)
Brett Wagner Mackenzie, MSc, Kristi Biswas, PhD, Michael W Taylor, PhD, Richard G Douglas, MD ChB, MD
Rationale: The role of the sinus microbiota in health and disease has not been well characterised, in part due to the overwhelming proportion of contaminating host DNA compared with recovered microbial DNA. A methods study examining different techniques for removing contaminating host DNA before metagenomic sequencing was conducted. We hypothesized that the enrichment techniques would improve recovery of microbial information from sinus swab samples.

Methods: Twelve samples were taken from each of three adult patients recruited from Auckland City Hospital, New Zealand who were undergoing functional endoscopic sinus surgery for chronic rhinosinusitis (CRS), an immune mediated disease characterised by chronic inflammation of the sinuses. Pairs of swabs from each patient were subject to three microbial DNA enrichment methods: 1. A series of centrifugation steps, followed by a standard DNA extraction, 2. A standard DNA extraction followed by enrichment of microbial DNA using NEBNext® Microbiome DNA Enrichment kit, and 3. Whole-genome amplification following the previous enrichment strategies.  Additionally, a no-treatment control and a whole-genome amplified control from each patient were sequenced. Paired-end shotgun metagenome sequencing was conducted on the Illumina HiSeq platform.

Results: Each enrichment method and the no-treatment controls were assessed for effectiveness based on the proportion of recovered microbial sequence reads to human-assigned sequences. Enrichment techniques improved the recovery of microbial sequences compared to the no-treatment controls (>99% of sequences belonged to host DNA).

Conclusions: We recommend enriching for microbial DNA when conducting metagenomic analyses on human-associated microbial communities. The appropriate enrichment technique depends on the research question and depth of sequencing required.