We found that standardized nomenclature is crucial for reducing ambiguity in microbiome studies. Specifically, we highlight the importance of distinguishing between "urinary bladder" samples obtained through catheterization or cystoscopic methods and "urogenital" samples collected via voided urine to account for potential contamination sources. Proper sample collection techniques are equally vital, with direct collection methods such as cystoscopy for urine or homogenized stool for fecal samples proving more reliable than alternative approaches.
Contamination prevention emerged as a key concern, particularly in low-biomass samples such as urine and saliva. We identified multiple contamination risks throughout the collection and processing workflow, emphasizing the necessity of sterile techniques, protective equipment, and rigorous decontamination protocols involving sodium hypochlorite and UV light. Contamination remains a persistent challenge despite these precautions, underscoring the need for enhanced quality control measures.
Sample storage conditions significantly influence microbiome integrity. While immediate freezing at -80°C is the gold standard, our analysis revealed that refrigeration at 4°C can preserve microbial diversity for fecal samples with minimal loss in composition. We also examined the efficacy of preservative buffers, noting that OMNIgene®GUT and AssayAssure performed well in stabilizing microbial communities when freezing was not feasible. However, we caution that preservatives can introduce biases, necessitating confirmational testing prior to use.
DNA extraction protocols further impact microbiome research outcomes, with variations in extraction kits affecting taxa composition and microbial diversity. Mechanical disruption, notably bead beating, was found to enhance microbial profiling by improving cell lysis and DNA yield. Additionally, sequencing approaches must be carefully selected based on study objectives. For urinary microbiota studies, primers such as V1V2 were more suitable than V4, which may underestimate species richness and be prone to human DNA contamination.
Our findings highlight the importance of methodological standardization while allowing flexibility for technological advancements (Fig 1). We recommend detailed documentation of all computational and laboratory procedures to enhance reproducibility. By implementing these guidelines, researchers can improve the reliability of microbiome studies and facilitate their translation into clinical applications.
Figure 1. Summary of best practice guidelines for microbiome research.
Written by: Ilaha Isali,1 Thomas R. Wong2
- Department of Urology, Weill Cornell Medicine/NewYork-Presbyterian Hospital
- School of Engineering, Case Western Reserve University
- I. Isali, T.R. Wong, S. Tian, Best Practice Guidelines for Collecting Microbiome Samples in Research Studies, Eur Urol Focus 10(6) (2024) 909-913.