Medical Scientist Training Program, University of California San Diego, La Jolla, California, USA.
Department of Pediatrics, University of California San Diego, La Jolla, California, USA.
mSystems. 2023 Feb 23;8(1):e0102922. doi: 10.1128/msystems.01029-22. Epub 2022 Dec 8.
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. 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.
可重复性是微生物组研究中一个既定的挑战,在样本收集到代码执行的各个阶段都存在多种因素。在这里,我们专注于尿生殖道微生物组分析中尿液样本的储存条件。我们使用从 10 名成年女性收集的尿液样本,研究了 AssayAssure Genelock(Genelock)在不同温度条件下对微生物组保存效果的影响,与无防腐剂的情况进行了比较。我们在 48 小时内改变了温度,以研究家庭自行收集尿液样本并运输到中央生物库过程中样本可能经历的条件的影响。研究了以下常见的实验室和运输条件:-20°C、环境温度、4°C、冻融循环和热循环。48 小时后,所有样本均在-80°C 下储存,直至处理。在使用高度敏感的 KatharoSeq 方案生成 16S rRNA 基因扩增子测序数据后,我们观察到个体间 alpha 和 beta 多样性指标的个体差异低于人际间差异,证实了其他样本类型个体微生物组变异性的报告。虽然在比较 Genelock 与无防腐剂时,beta 多样性没有显著差异,但与在更冷的温度(-20°C 和 4°C)下运输的 Genelock 样本相比,在没有防腐剂的情况下在-20°C 下运输的样本具有更高的一致性。我们的结果表明,Genelock 在一系列温度下使用时不会引入大量微生物偏倚,并且在较冷温度下效果最佳。尿生殖道微生物组是一个研究不足但很重要的人类微生物组生态位。相对较新的发现——尿液并非无菌,这一发现激发了相关研究。尿液中的微生物与健康问题有关,包括尿路感染、尿失禁和癌症。仅尿路感染和急迫性尿失禁在美国每年就分别花费 35 亿美元和 826 亿美元。鉴于尿液的生物量低、该领域的新颖性以及有限的重现性证据,研究尿液样本储存条件以优化科学严谨性至关重要。高效可靠的保存方法为家庭自行收集和运输提供了信息,增加了在更大规模研究中的潜在用途。在这里,我们研究了尿生殖道样本 16S rRNA 基因扩增子测序结果中缓冲液和温度变化的影响,提供了常见储存方法对尿生殖道微生物组结果的影响的数据。