Comparison of fecal collection methods on variation in gut metagenomics and untargeted metabolomics

Huihui Guan, Yanni Pu, Chenglin Liu, Tao Lou, Shishang Tan, Mengmeng Kong, Zhonghan Sun, Zhendong Mei, Qibin Qi, Zhexue Quan, Guoping Zhao, Yan Zheng

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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 280°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 a- and b-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.

Original languageEnglish (US)
Article numbere00636-21
Issue number5
StatePublished - Oct 2021


  • Collection method
  • Feces
  • Metabolomics
  • Metagenomics
  • Reliability
  • Wholegenome shotgun sequencing

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology


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