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Related Experiment Video

Updated: Apr 5, 2026

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
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Methods for Improving Human Gut Microbiome Data by Reducing Variability through Sample Processing and Storage of

Monika A Gorzelak1, Sandeep K Gill1, Nishat Tasnim1

  • 1Department of Biology, University of British Columbia Okanagan, Kelowna, BC, V1V 1V7, Canada.

Plos One
|August 8, 2015
PubMed
Summary

Properly processing and storing human stool samples is crucial for accurate gut microbiome analysis. Freezing samples quickly, storing them for under three days, and homogenizing them minimizes data variability.

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Area of Science:

  • Microbiology
  • Genetics
  • Bioinformatics

Background:

  • Gut microbiome analysis is vital for understanding diseases like IBD, obesity, and diabetes.
  • Standardized sampling methods are often overlooked in human microbiome research.
  • Sample preparation, handling, and storage significantly impact microbiome data outcomes.

Purpose of the Study:

  • To investigate how sample preparation, handling, and storage affect DNA-based gut microbiome analysis.
  • To identify optimal protocols for processing and storing human stool samples to reduce data variability.
  • To provide practical solutions for improving the reliability of human microbiome research.

Main Methods:

  • Assessed the impact of stool subsampling, homogenization, and storage conditions (room temperature, freezer, freeze-thawing) on bacterial taxa.
  • Utilized quantitative PCR (qPCR) for DNA-based bacterial community analysis.
  • Compared results from samples stored using different methods, including RNAlater.

Main Results:

  • Stool subsampling introduced significant variability; homogenization reduced this intra-sample variability.
  • Bacterial taxa changed with storage beyond 15 minutes at room temperature and 3 days in a frost-free freezer.
  • Freeze-thawing affected abundance beyond 4 cycles; RNAlater reduced DNA yield and bacterial detection.

Conclusions:

  • Recommend freezing stool within 15 minutes of defecation.
  • Advise storage in a domestic frost-free freezer for less than 3 days.
  • Emphasize homogenizing stool samples before DNA extraction to enhance microbiome data reliability.