Effects of Variation in Urine Sample Storage Conditions on 16S Urogenital Microbiome Analyses

Abstract Replicability is a well-established challenge in microbiome research with a variety of contributing factors at all stages, from sample collection to code execution. Here, we focus on voided urine sample storage conditions for urogenital microbiome analysis. Using urine samples collected from 10 healthy adult women, we investigated the microbiome preservation efficacy of AssayAssure® Genelock (as opposed to no preservative) under different temperature conditions. We varied temperature over 48 hours in order to examine the impact of conditions samples may experience with home voided urine collection and shipping to a central biorepository. The following common lab and shipping conditions were investigated: -20C, ambient temperature, 4C, a freeze-thaw cycle, and a heat cycle. At 48 hours, all samples were stored at -80C until processing. After generating 16S rRNA gene amplicon sequencing data using the highly sensitive KatharoSeq protocol, we observed individual variation in both alpha and beta diversity metrics below interhuman differences, corroborating reports of individual microbiome variability in other specimen types. While there was no significant difference in beta diversity when comparing AssayAssure® Genelock vs. no preservative, we did observe a higher concordance with AssayAssure samples shipped at colder temperatures (−20C and 4C) when compared to the samples shipped at -20C without preservative. Our results indicate that AssayAssure does not introduce a significant amount of microbial bias when used on a range of temperatures but is most effective at colder temperatures. Importance The urogenital microbiome is an understudied yet important human microbiome niche. Research has been stimulated by the relatively recent discovery that urine is not sterile: urinary tract microbes have been linked to health problems including urinary infections, incontinence, and cancer. The quality of life and economic impact of UTIs and urgency incontinence alone are enormous, with $3.5 billion and $82.6 billion respectively spent in the U.S. annually. Given the low biomass of urine, novelty of the field, and well-established replicability bias in microbiome studies, it is critical to study storage conditions on urine samples to minimize microbial biases. Efficient and reliable preservation methods permit home self-sample collection and shipping, increasing the accessibility of larger-scale studies. Here, we examined both buffer and temperature variation effects on 16S rRNA gene amplicon sequencing results from urogenital samples, providing data on the consequences of common storage methods on urogenital microbiome results.