New technologies like next-generation sequencing have led to a proliferation of studies investigating the role of the gut microbiome in human health, particularly population-based studies that rely upon participant self-collection of samples. However, the impact of methodological differences in sample shipping, storage, and processing are not well-characterized for these types of studies, especially when transit times may exceed 24 h. The aim of this study was to experimentally assess microbiota stability in stool samples stored at 4 °C for durations of 6, 24, 48, 72, and 96 h with no additives to better understand effects of variable shipping times in population-based studies. These data were compared to a baseline sample that was immediately stored at − 80 °C after stool production. Compared to the baseline sample, we found that the alpha-diversity metrics Shannon’s and Inverse Simpson’s had excellent intra-class correlations (ICC) for all storage durations. Chao1 richness had good to excellent ICC. We found that the relative abundances of bacteria in the phyla Verrucomicrobia, Actinobacteria, and Proteobacteria had excellent ICC with baseline for all storage durations, while Firmicutes and Bacteroidetes ranged from moderate to good. We interpreted the ICCs as follows: poor: ICC < 0.50, moderate: 0.50 < ICC < 0.75, good: 0.75 < ICC < 0.90, and excellent: ICC > 0.90. Using the Bray–Curtis dissimilarity index, we found that the greatest change in community composition occurred between 0 and 24 h of storage, while community composition remained relatively stable for subsequent storage durations. Samples showed strong clustering by individual, indicating that inter-individual variability was greater than the variability associated with storage time. The results of this analysis suggest that several measures of alpha diversity, relative abundance, and overall community composition are robust to storage at 4 °C for up to 96 h. We found that the overall community richness was influenced by storage duration in addition to the relative abundances of sequences within the Firmicutes and Bacteroidetes phyla. Finally, we demonstrate that inter-individual variability in microbiota composition was greater than the variability due to changing storage durations.

中文翻译：

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评估储存时间对粪便微生物群丰富度、多样性和组成稳定性的影响

新一代测序等新技术导致了大量研究肠道微生物组在人类健康中的作用，特别是依赖于参与者自我收集样本的基于人群的研究。然而，在这些类型的研究中，样品运输、储存和加工方法学差异的影响并没有得到很好的表征，尤其是在运输时间可能超过 24 小时的情况下。本研究的目的是通过实验评估在 4 °C 下储存 6、24、48、72 和 96 小时且无添加剂的粪便样本中微生物群的稳定性，以更好地了解不同运输时间在基于人群的研究中的影响。将这些数据与粪便产生后立即储存在 - 80 °C 的基线样本进行比较。与基线样本相比，我们发现，Shannon 和 Inverse Simpson 的 alpha 多样性指标在所有存储期间都具有出色的类内相关性 (ICC)。Chao1 丰富度有好到优秀的 ICC。我们发现疣微菌门、放线菌门和变形菌门中细菌的相对丰度在所有储存期限内都具有出色的 ICC 和基线，而厚壁菌门和拟杆菌门的范围从中等到良好。我们对 ICC 的解释如下：差：ICC < 0.50，中等：0.50 < ICC < 0.75，好：0.75 < ICC < 0.90，和优秀：ICC > 0.90。使用 Bray-Curtis 相异指数，我们发现群落组成的最大变化发生在储存 0 到 24 小时之间，而群落组成在随后的储存持续时间内保持相对稳定。样本显示出强烈的个体聚类，表明个体间变异性大于与储存时间相关的变异性。该分析的结果表明，α 多样性、相对丰度和整体群落组成的几种度量对于在 4°C 下储存长达 96 小时是稳健的。我们发现，除了厚壁菌门和拟杆菌门内序列的相对丰度外，总体群落丰富度还受储存持续时间的影响。最后，我们证明了微生物群组成的个体间变异性大于由于储存持续时间变化引起的变异性。和整体群落组成对于在 4°C 下储存长达 96 小时是稳健的。我们发现，除了厚壁菌门和拟杆菌门内序列的相对丰度外，总体群落丰富度还受储存持续时间的影响。最后，我们证明了微生物群组成的个体间变异性大于由于储存持续时间变化引起的变异性。和整体群落组成对于在 4°C 下储存长达 96 小时是稳健的。我们发现，除了厚壁菌门和拟杆菌门内序列的相对丰度外，总体群落丰富度还受储存持续时间的影响。最后，我们证明了微生物群组成的个体间变异性大于由于储存持续时间变化引起的变异性。