ena-STUDY-UNIVERSITY OF AUCKLAND-23-05-2017-01:52:21:119-334
Assessment of Microbial DNA Enrichment Techniques from Sino-Nasal Swab Samples for Metagenomics
The role of the sinus microbiota, including bacteria, fungi and viruses, in health and disease remains unclear despite the application of molecular microbiological techniques to describe the microbiome. This is due, in part, to the overwhelming proportion of contaminating host DNA compared with recovered microbial DNA. In this study, three techniques were assessed for microbial DNA enrichment: 1. A series of centrifugation steps, 2. Enrichment of microbial DNA using the NEBNext® Microbiome DNA Enrichment kit, and 3. Whole-genome amplification following the previous enrichment strategies. A no-treatment control and a whole-genome amplified control were also included. Swab samples from three adult patients undergoing functional endoscopic sinus surgery for chronic rhinosinusitis (CRS) were collected intraoperatively for this study. Paired-end shotgun metagenome sequencing was conducted using Illumina HiSeq and Bacteria-targeted 16S rRNA gene sequencing was conducted on Illumina MiSeq to assess bacterial community composition. After quality filtering of obtained sequences, the centrifugation method returned the highest proportion of microbial reads (1.1 ± 1.7%) compared to the no-treatment control (0.15 ± 0.07%). However, this result was neither reproducible nor was centrifugation significantly different to the other methods. Despite continued low recovery of total microbial DNA from metagenomic sequencing, a Propionibacterium acnes genome (97% complete, 0% contamination) was recovered, suggesting metagenomic sequencing techniques can still be successfully applied to investigate the microbial component of CRS. Based on the results from this study, we recommend omitting microbial DNA enrichment steps and sequencing fewer samples per metagenomic sequencing run to increase the depth of sequencing without altering in situ microbial community structure.
The role of the sinus microbiota, including bacteria, fungi and viruses, in health and disease remains unclear despite the application of molecular microbiological techniques to describe the microbiome. This is due, in part, to the overwhelming proportion of contaminating host DNA compared with recovered microbial DNA. In this study, three techniques were assessed for microbial DNA enrichment: 1. A series of centrifugation steps, 2. Enrichment of microbial DNA using the NEBNext® Microbiome DNA Enrichment kit, and 3. Whole-genome amplification following the previous enrichment strategies. A no-treatment control and a whole-genome amplified control were also included. Swab samples from three adult patients undergoing functional endoscopic sinus surgery for chronic rhinosinusitis (CRS) were collected intraoperatively for this study. Paired-end shotgun metagenome sequencing was conducted using Illumina HiSeq and Bacteria-targeted 16S rRNA gene sequencing was conducted on Illumina MiSeq to assess bacterial community composition. After quality filtering of obtained sequences, the centrifugation method returned the highest proportion of microbial reads (1.1 ± 1.7%) compared to the no-treatment control (0.15 ± 0.07%). However, this result was neither reproducible nor was centrifugation significantly different to the other methods. Despite continued low recovery of total microbial DNA from metagenomic sequencing, a Propionibacterium acnes genome (97% complete, 0% contamination) was recovered, suggesting metagenomic sequencing techniques can still be successfully applied to investigate the microbial component of CRS. Based on the results from this study, we recommend omitting microbial DNA enrichment steps and sequencing fewer samples per metagenomic sequencing run to increase the depth of sequencing without altering in situ microbial community structure.