Human Phenome Institute, School of Life Sciences, Fudan Universitygrid.8547.e, Shanghai, China.
MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan Universitygrid.8547.e, Shanghai, China.
mSphere. 2021 Oct 27;6(5):e0063621. doi: 10.1128/mSphere.00636-21. Epub 2021 Sep 15.
Integrative analysis of high-quality metagenomics and metabolomics data from fecal samples provides novel clues for the mechanism underpinning gut microbe-human interactions. However, data regarding the influence of fecal collection methods on both metagenomics and metabolomics are sparse. Six fecal collection methods (the gold standard [GS] [i.e., immediate freezing at -80°C with no solution], 95% ethanol, RNAlater, OMNIgene Gut, fecal occult blood test [FOBT] cards, and Microlution) were used to collect 88 fecal samples from eight healthy volunteers for whole-genome shotgun sequencing (WGSS) and untargeted metabolomic profiling. Metrics assessed included the abundances of predominant phyla and α- and β-diversity at the species, gene, and pathway levels. Intraclass correlation coefficients (ICCs) were calculated for microbes and metabolites to estimate (i) stability (day 4 versus day 0 within each method), (ii) concordance (day 0 for each method versus the GS), and (iii) reliability (day 4 for each method versus the GS). For the top 4 phyla and microbial diversity metrics at the species, gene, and pathway levels, generally high stability and reliability were observed for most methods except for 95% ethanol; similar concordances were seen for different methods. For metabolomics data, 95% ethanol showed the highest stability, concordance, and reliability (median ICCs = 0.71, 0.71, and 0.65, respectively). Taken together, OMNIgene Gut, FOBT cards, RNAlater, and Microlution, but not 95% ethanol, were reliable collection methods for gut metagenomic studies. However, 95% ethanol was the best for preserving fecal metabolite profiles. We recommend using separate collecting methods for gut metagenomic sequencing and fecal metabolomic profiling in large population studies. The choice of fecal collection method is essential for studying gut microbe-human interactions in large-scale population-based research. In this study, we examined the effects of fecal collection methods and storage time at ambient temperature on variations in the gut microbiome community composition; microbial diversity metrics at the species, gene, and pathway levels; antibiotic resistance genes; and metabolome profiling. Our findings suggest using different fecal sample collection methods for different data generation purposes. OMNIgene Gut, FOBT cards, RNAlater, and Microlution, but not 95% ethanol, were reliable collection methods for gut metagenomic studies. However, 95% ethanol was the best for preserving fecal metabolite profiles.
综合分析粪便样本的高质量宏基因组学和代谢组学数据为肠道微生物与人体相互作用的机制提供了新的线索。然而，关于粪便采集方法对宏基因组学和代谢组学的影响的数据还很匮乏。本研究使用了六种粪便采集方法（金标准[即立即在-80°C 下冷冻，不使用任何溶液]、95%乙醇、RNAlater、OMNIgene Gut、粪便潜血检测[FOBT]卡和 Microlution）从 8 名健康志愿者中采集了 88 份粪便样本，用于全基因组鸟枪法测序（WGSS）和非靶向代谢组学分析。评估的指标包括主要菌群的丰度以及在物种、基因和途径水平上的α-和β-多样性。计算了微生物和代谢物的组内相关系数（ICC），以评估（i）稳定性（每种方法在第 4 天和第 0 天之间），（ii）一致性（每种方法在第 0 天与金标准之间），和（iii）可靠性（每种方法在第 4 天与金标准之间）。对于前 4 种菌群和微生物多样性指标在物种、基因和途径水平上，除 95%乙醇外，大多数方法均表现出较高的稳定性和可靠性；不同方法之间的一致性相似。对于代谢组学数据，95%乙醇表现出最高的稳定性、一致性和可靠性（中位数 ICC 分别为 0.71、0.71 和 0.65）。总之，OMNIgene Gut、FOBT 卡、RNAlater 和 Microlution，但不是 95%乙醇，是肠道宏基因组研究中可靠的采集方法。然而，95%乙醇是保存粪便代谢物谱的最佳方法。我们建议在大型人群研究中，将肠道宏基因组测序和粪便代谢组学分析分开使用不同的采集方法。在这项研究中，我们研究了粪便采集方法和在室温下储存时间对肠道微生物群落组成变化、物种、基因和途径水平的微生物多样性指标、抗生素耐药基因和代谢组学分析的影响。我们的研究结果表明，在大规模基于人群的研究中，为了研究肠道微生物与人体的相互作用，需要选择不同的粪便采集方法。OMNIgene Gut、FOBT 卡、RNAlater 和 Microlution，但不是 95%乙醇，是肠道宏基因组研究中可靠的采集方法。然而，95%乙醇是保存粪便代谢物谱的最佳方法。
