Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics.
Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics.
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DOI：
10.1128/msphere.00636-21
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发表时间：
2021-10-27
期刊：
影响因子：
4.8
通讯作者：
Zheng Y
中科院分区：
文献类型：
--
作者：
Guan H;Pu Y;Liu C;Lou T;Tan S;Kong M;Sun Z;Mei Z;Qi Q;Quan Z;Zhao G;Zheng Y
对粪便样本的高质量宏基因组学和代谢组学数据进行综合分析，为肠道微生物与人类相互作用的基础机制提供了新的线索。然而，有关粪便收集方法对宏基因组学和代谢组学影响的数据很少。使用六种粪便收集方法（黄金标准 [GS] [即 -80°C 立即冷冻，无溶液]、95% 乙醇、RNAlater、OMNIgene Gut、粪便潜血测试 [FOBT] 卡和 Microlution）从 8 名健康志愿者收集 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门的丰度以及物种、基因和途径水平的α和β多样性。计算微生物和代谢物的组内相关系数 (ICC)，以评估 (i) 稳定性（每种方法的第 4 天与第 0 天），(ii) 一致性（每种方法的第 0 天与 GS），以及 (iii) 可靠性（每种方法的第 4 天与 GS）。对于物种、基因和途径水平上的前 4 个门和微生物多样性指标，除 95% 乙醇外，大多数方法总体上观察到较高的稳定性和可靠性；不同的方法也有类似的一致性。对于代谢组学数据，95%乙醇显示出最高的稳定性、一致性和可靠性（中位ICC分别=0.71、0.71和0.65）。综上所述，OMNIgene Gut、FOBT 卡、RNAlater 和 Microlution（而非 95% 乙醇）是肠道宏基因组研究的可靠收集方法。然而，95% 乙醇是保存粪便代谢物特征的最佳选择。我们建议在大群体研究中使用单独的收集方法进行肠道宏基因组测序和粪便代谢组学分析。重要性 在大规模人群研究中，粪便收集方法的选择对于研究肠道微生物与人类的相互作用至关重要。在这项研究中，我们研究了粪便收集方法和环境温度下的储存时间对肠道微生物群落组成变化的影响；物种、基因和途径水平的微生物多样性指标；抗生素抗性基因；和代谢组分析。我们的研究结果表明，针对不同的数据生成目的，使用不同的粪便样本收集方法。 OMNIgene Gut、FOBT 卡、RNAlater 和 Microlution，但不是 95% 乙醇，是肠道宏基因组研究的可靠收集方法。然而，95% 乙醇是保存粪便代谢物特征的最佳选择。
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. IMPORTANCE 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.