Systematic Analysis of Impact of Sampling Regions and Storage Methods on Fecal Gut Microbiome and Metabolome Profiles.
Author(s): Liang Y, Dong T, Chen M, He L, Wang T, Liu X, Chang H, Mao JH, Hang B, Snijders AM, Xia Y
Publication: mSphere, 2020, Vol. 5, Page
PubMed ID: 31915218 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of preservation method, storage temperature, thawing method, and sampling location on microbiome and metabolome profiles of fecal specimens.
Conclusion of Paper
No significant differences were observed in the microbial alpha diversity index, beta diversity index, or in relative microbial abundance based on sampling location (head, body, or tail), subsampling location (surface, core, or combined core and surface), storage temperature (liquid nitrogen, household freezer, room temperature), or thawing method (on ice or without ice), and differences in relative microbial diversity between preservation methods were smaller than those found between patients. Similarly, differences in metabolite levels between sampling and subsampling location were attributed to inter-individual variability. However, five of the 176 metabolites were consistently downregulated for all three participants in specimens stored in RNAlater compared to those stored at room temperature or frozen.
Studies
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Study Purpose
This study investigated the effects of preservation method, storage temperature, thawing method, and sampling location on microbiome and metabolome profiles of fecal specimens. Stool specimens were collected in a prepared sterile enamel tray from three healthy 34-month-old study participants in their home. Specimens were divided equally along the longitudinal axis. To evaluate the effects of storage and thawing conditions, half was homogenized immediately and four aliquots were collected for each preservation/storage method (two 200-mg aliquots for microbiome analysis and two 100-mg aliquots for metabolomics analysis). Aliquots were preserved using seven different methods: snap-freezing in liquid nitrogen for 52 h (control); freezing in a common household freezer (-16°C) for 48 h; freezing in RNALater in a household freezer (-16°C) for 48 h; storage at room temperature for 52 h (48 h plus 4 h) with or without RNALater. To investigate the effects of thawing method, specimens stored in household freezer with or without RNA later for 48 h were thawed at room temperature for 4 h either gradually (on ice) or fast (no ice). To investigate differences between sampling regions, the other half of each stool specimen was separated into three equal sections according to the order of defecation (head, body, and tail) and four aliquots (two 200-mg aliquots for microbiome analysis and two 100-mg aliquots for metabolomics analysis) were collected from the surface, core, and a combination of surface and core. DNA was extracted from all specimens using the Qiagen Stool Minikit according to the manufacturer’s instructions. PCR amplification of the bacterial hypervariable V3 region of the 16S rRNA gene was performed. Sequencing libraries were generated using the Illumina TruSeq DNA PCR-Free Library Preparation Kit and the library was sequenced on an Illumina HiSeq platform. For metabolite analysis, specimens were homogenized by vortexing for 15 sec and centrifuged at 14,000 x g for 15 min. Supernatants were filtered through a 22 µm membrane, dried, reconstituted in deionized water, and then analyzed by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS).
Summary of Findings:
Principal-component analysis (PCA) revealed that individual variability was responsible for the majority of microbial diversity (P<0.001) and no significant differences were observed compared to control specimens in either the alpha diversity index, beta diversity index, or in relative abundance based on sampling location (head, body, or tail), subsampling location (surface, core, or combined core and surface), preservation method (RNAlater, freezing etc), storage temperature, or thawing method. No differences in relative microbial abundance were found in specimens stored at room temperature or those frozen and subjected to gradual thawing or fast thawing compared to control specimens snap-frozen in liquid nitrogen, but levels of Bacteroidales decreased and Clostridiales levels increased with storage in RNAlater, but inter-individual variability was very high. While levels of 22 of the 176 metabolites analyzed varied significantly across different sampling locations and levels for two metabolites differed between subsample locations and the homogenized control specimens, PCA revealed that these differences were due to inter-individual variability. A total of 21 metabolites were significantly downregulated and 22 metabolites were significantly upregulated in specimens stored in RNALater compared to those stored at room temperature or frozen (P<0.05, all); five of which (5-hydroxylysine, deoxyinosine, glucosamine 6-phosphate, L-lysine, and ribothymidine) were consistently downregulated for all three participants (P<0.05).
Biospecimens
Preservative Types
- Frozen
- RNAlater
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Small molecule HPLC-MS DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Head of fecal specimen
Body of fecal specimen
Tail of fecal specimen
Core of fecal specimen
Surface of fecal specimen
Combined surface and core of fecal specimen
Biospecimen Preservation Type of fixation/preservation Frozen
Snap frozen
RNAlater
Storage Storage temperature Room Temperature
Liquid nitrogen
-16°C
Storage Thaw temperature/condition Room temperature for 4 hours on ice
Room temperature for 4 hours without ice
Biospecimen Aliquots and Components Biospecimen heterogeneity Biospecimen core
Biospecimen periphery