Stamper Christopher E, Hoisington Andrew J, Ellis Joseph C, Lowry Christopher A, Brenner Lisa A
Veterans Health Administration, Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC) for Veteran Suicide Prevention, Rocky Mountain Regional Veterans Affairs Medical Center (RMRVAMC), Aurora, Colorado, United States of America.
Department of Physical Medicine and Rehabilitation, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
PLoS One. 2025 Aug 13;20(8):e0324351. doi: 10.1371/journal.pone.0324351. eCollection 2025.
Over the past decade, studies have been conducted to increase the understanding of associations between the fecal microbiome and human health. In conjunction, researchers have investigated the effects of study design, methods, molecular processing, and sequencing techniques. However, a lack of standardization of fecal sample collection methodology has introduced heterogeneity in sequencing results. Sources of variability include sample collection methods, storage temperatures, and transport times. Here we present 16S rRNA gene amplicon sequencing results from two sample collection methods (unstabilized sterile swab and stabilized OmniGene Gut Kits) collected from the same fecal specimens. The paired samples were collected either at the research facility or the participants' home and ground shipped to the research facility at ambient temperature. Therefore, samples were exposed to variable temperatures and transport times. We found that fecal sample collection methods resulted in taxonomic and diversity differences that showed distinct patterns between swab and OmniGene samples. Swab samples were disproportionally affected by increased transport time, but differences in taxa and diversity were driven more by sample collection method, as compared to transport time. Based on previous studies, many of the taxa that were associated with sample collection methods and transport times have clinical relevance. Collectively, this research highlights: 1) the need for further standardization of methods for fecal microbiome studies; 2) limitations of direct comparisons between different fecal sample collection methods; and 3) the importance of careful consideration of sample collection methods for future studies and meta-analyses.
在过去十年中，人们开展了多项研究，以增进对粪便微生物群与人类健康之间关联的理解。与此同时，研究人员还调查了研究设计、方法、分子处理和测序技术的影响。然而，粪便样本采集方法缺乏标准化，导致测序结果存在异质性。变异来源包括样本采集方法、储存温度和运输时间。在此，我们展示了从同一粪便标本中采用两种样本采集方法（未稳定化的无菌拭子和稳定化的全基因肠道试剂盒）获得的16S rRNA基因扩增子测序结果。配对样本在研究机构或参与者家中采集，并在室温下通过地面运输送至研究机构。因此，样本暴露于不同的温度和运输时间。我们发现，粪便样本采集方法导致了分类学和多样性差异，拭子样本和全基因样本之间呈现出不同的模式。拭子样本受运输时间增加的影响不成比例，但与运输时间相比，分类群和多样性的差异更多地由样本采集方法驱动。根据以往研究，许多与样本采集方法和运输时间相关的分类群具有临床相关性。总体而言，这项研究强调了：1）粪便微生物群研究方法需要进一步标准化；2）不同粪便样本采集方法之间直接比较的局限性；3）在未来研究和荟萃分析中仔细考虑样本采集方法的重要性。
