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Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
Published on: March 19, 2018
Human faecal collection methods demonstrate a bias in microbiome composition by cell wall structure
Emma-Jane Watson1, Jennifer Giles1, Benjamin L Scherer1
1CSIRO Health and Biosecurity, Gate 13 Kintore Avenue, Adelaide, South Australia, 5000, Australia.
Abstract:
Clinical trial faecal collections present challenges through geographical spread and inexperienced participants. Collection techniques have been developed and tested to overcome these challenges, but previous studies investigating these techniques have demonstrated a highly variable capacity for sample preservation. Furthermore, these studies typically only examine either preservation of genetic content or metabolites, not both. This study investigated the Stool Nucleic Acid Collection and Preservation Tube (Norgen BioTek Corp) for the preservation of both microbial DNA and microbial organic acid metabolites in human faecal samples when compared to frozen samples. Twenty six healthy adult participants were instructed to collect a bowel movement, subsample into collection tubes and immediately transfer the remaining bulk to -20 °C storage. Resulting organic acid concentrations remained comparable across methods when the preservation tubes were used correctly. The 16S rRNA gene sequencing data revealed twenty significantly different bacterial genera between the two collection methods. Ten Gram-negative genera were more abundant in the collection tubes, and ten Gram-positive genera were more abundant in the fresh frozen samples. This study has illustrated that faecal collection methods bias the microbial community profile according to Gram status and this should be considered when designing studies that collect and store human faecal samples.
Insights
This study compared a novel stool collection tube to freezing for preserving fecal samples. While organic acid metabolites were comparable, the collection tube method altered the microbial community profile, favoring Gram-negative bacteria.
Area of Science:
- Microbiology
- Biotechnology
- Clinical Trials
Background:
- Clinical trial fecal sample collection faces challenges like participant variability and geographical dispersion.
- Existing sample preservation techniques show inconsistent results and often assess only DNA or metabolites, not both.
- Accurate fecal sample preservation is crucial for reliable microbiome and metabolomic research.
Purpose of the Study:
- To evaluate the efficacy of the Stool Nucleic Acid Collection and Preservation Tube (Norgen BioTek Corp) for preserving both microbial DNA and organic acid metabolites in human fecal samples.
- To compare the preservation capabilities of the collection tubes against traditional frozen storage (-20°C).
- To identify potential biases introduced by the collection method on microbial community composition and metabolite profiles.
Main Methods:
- Twenty-six healthy adults collected fecal samples, with subsamples placed in Norgen BioTek collection tubes and the remainder immediately frozen at -20°C.
- Organic acid concentrations were analyzed to assess metabolite preservation.
- 16S rRNA gene sequencing was performed to compare microbial DNA profiles between the two collection methods.
Main Results:
- Organic acid concentrations were comparable between samples stored in collection tubes and fresh frozen samples when tubes were used correctly.
- Significant differences in bacterial genera were observed between the two collection methods using 16S rRNA gene sequencing.
- Gram-negative bacterial genera were more abundant in samples from collection tubes, while Gram-positive genera were more abundant in fresh frozen samples.
Conclusions:
- The Stool Nucleic Acid Collection and Preservation Tube can preserve organic acid metabolites effectively but introduces a bias in the microbial community profile, favoring Gram-negative bacteria.
- The observed bias based on Gram status highlights the importance of considering collection and storage methods in study design.
- Researchers must account for potential biases introduced by fecal collection techniques to ensure accurate interpretation of microbiome data.
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