Optimization of DNA extraction from human urinary samples for mycobiome community profiling

Area of Science:

• Microbiology
• Genomics

Background:

• The human urinary tract harbors a diverse microbial community, even in healthy individuals.
• Culture-independent methods like next-generation sequencing (NGS) offer broad insights into these communities, including fungi.
• A key challenge is isolating DNA representative of the entire microbial community, especially fungi.

Purpose of the Study:

• To evaluate and optimize DNA extraction protocols for comprehensive profiling of the urinary tract microbiome.
• To improve the yield and representation of both fungal and bacterial DNA from urine samples.
• To compare an optimized protocol against commercial methods for microbial community analysis.

Main Methods:

• Evaluated modifications to standard DNA extraction procedures using standardized urine samples.
• Quantified DNA yields (overall, fungal, bacterial) using quantitative PCR.
• Combined optimized steps including enzymatic, mechanical, and thermal disruption, proteinase K treatment, and carrier DNA use.
• Compared the optimized protocol with commercial kits using NGS for community representation and diversity.

Main Results:

• Multiple disruption steps (enzymatic, mechanical, thermal, proteinase K) significantly increased DNA yields.
• Optimized protocol with low-speed centrifugation for concentrating samples enhanced microbial representation and diversity.
• The optimized protocol demonstrated superior fungal DNA yield and representation compared to other methods.
• NGS analysis confirmed equivalent representation of bacterial populations with the optimized protocol.

Conclusions:

• Modifications in urine storage, preparation, and DNA extraction significantly improve microbial community profiling via NGS.
• The developed optimized protocol enhances fungal and bacterial DNA extraction from urine.
• This technique enables concurrent evaluation of both bacterial and fungal populations in urine samples using NGS.