Evaluation of Urine Bacterial DNA Isolation Protocols for Sequencing Analysis of the Urinary Microbiome
Matthew J. Moynihan, MD, MPH; Travis Sullivan, MS; Kimberly Rieger-Christ, PhD
Lahey Clinic, Burlington, MA
BACKGROUND: The influence of the human bacterial microbiome on a myriad of diseases has been recently elucidated, but limited data are available regarding its role in genitourinary pathophysiology. Research into the urinary microbiome is limited in part by technical challenges and ease of contamination. If a better understanding of the urinary microbiome and its relationship to genitourinary diseases is to be gained, optimization of analytic techniques and practices needs to be undertaken. This pilot study sought to compare preanalytical treatment of urine and currently available DNA isolation kits in the analysis of the urinary microbiome.
METHODS: Clean catch urine samples were obtained from patients under an IRB approved study. Treatment of the urine was divided into two groups: storage at -80℃ or fresh urine. Samples were sterilized by centrifugation and ultrafiltration and then spiked with either a known microbial standard (Zymo Research) or a control solution (phosphate buffer saline). Three different commercially available DNA isolation kits (Zymo Research, MoBio, and Qiagen) were used to extract DNA from samples. Twenty two samples were analyzed by 16S rRNA PCR/Illumina MiSeq gene sequencing in order to ascertain the best isolation method. Gene sequencing allowed for identification and determination of relative abundance of bacterial genera amongst the groups as compared to the known microbial standard.
RESULTS: Weighted UniFrac analysis of sample beta diversity demonstrated different kits have an effect on observed microbiome (p =0.064), and frozen samples are not as consistent as fresh samples (p=0.023). Determination of genera amongst samples revealed a disproportionately elevated relative abundance of staphylococcus species. The alpha diversity analysis to determine a measurement of microbial richness and evenness within a sample showed that a modified version of the Qiagen protocol was most closely consistent with the known microbial standard.
CONCLUSIONS: This study found that the specific DNA isolation kit as well as the pre-analysis treatment of the sample had an impact on the observed microbiome. The inherent low bacterial DNA biomass of urine samples proves to be a challenging feature of urinary microbiome analysis. Risk of sample contamination at any point in the process, from sample collection to final product, demands scrutiny in interpretation of low biomass samples. Future research involving the urinary microbiome should seek to minimize pre-analysis sample treatment differences between specimens. If using a commercially available bacterial DNA isolation kit, researchers should utilize a protocol such as the Qiagen kit with modifications that most closely represents a known microbial standard, especially given the setting of the technically challenging urine sample.
Back to 2018 Program