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 adult females, we investigated the microbiome preservation efficacy of AssayAssure Genelock (Genelock), compared with no preservative, under different temperature conditions. We varied temperature over 48 h 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: -20°C, ambient temperature, 4°C, freeze-thaw cycle, and heat cycle. At 48 h, all samples were stored at -80°C 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 Genelock versus no preservative, we did observe a higher concordance with Genelock samples shipped at colder temperatures (-20°C and 4°C) when compared with the samples shipped at -20°C without preservative. Our results indicate that Genelock does not introduce a significant amount of microbial bias when used on a range of temperatures and 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 United States. annually. Given the low biomass of urine, novelty of the field, and limited reproducibility evidence, it is critical to study urine sample storage conditions to optimize scientific rigor. Efficient and reliable preservation methods inform methods for home self-sample collection and shipping, increasing the potential use in 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.

Keywords: 16S; microbiome; sample storage; urobiome; urogenital microbiome.

FIG 1

Experimental design and clustering by individual. (A) 10 healthy adult females gave a single 10-mL urine sample, which was processed in triplicate and subject to varying temperature conditions with the presence or absence of AssayAssure Genelock (Genelock). This figure was created with BioRender.com. (B) Principal-coordinate analysis plots of weighted and unweighted UniFrac distances.

FIG 2

Quantification of results. Effect of temperature treatment and presence of Genelock on urogenital microbiome composition. Genelock is present in all samples other than the comparator group, which was stored at −20°C with no preservative. (A) Distances between different temperature treatments and comparator group unweighted and weighted UniFrac, BrayCurtis, and Jaccard distances. Interhuman distance was calculated by calculating the distance between each individual’s samples to the other individual’s samples (distances between samples from the same individual were not taken into account) and taking the median distance. Interreplicate distance was calculated by finding the distance between replicates (replicates are samples from the same individual that were treated with the same preservative method and stored at the same temperature) and taking the median replicate distance. (B) Shannon and faith PD alpha diversity differences between different temperature treatments and the comparator group. (C) Values are based on permutation tests of variance (stepwise ANOVA). (D) ADONIS (a multivariate analysis of variance) test. For this test Genelock samples stored at −20°C were compared against the comparator group samples (–20°C; no preservative) to capture the effect of the individual and preservative.