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Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
Published on: March 19, 2018
Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence
Gary D Wu1, James D Lewis, Christian Hoffmann
1Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076 USA. gdwu@mail.med.upenn.edu
BMC Microbiology
|August 3, 2010
Summary
Analyzing the human gut microbiome requires optimal methods. This study compared fecal sample processing techniques, finding that DNA purification significantly impacts results, especially for bacterial abundance analysis.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- The human gut microbiome plays a crucial role in health and disease.
- Optimal methods for analyzing fecal bacterial communities are still being developed.
- 16S rRNA gene sequencing is a common method for surveying microbial communities.
Purpose of the Study:
- To compare different methods for processing human fecal samples for 16S rRNA gene sequencing.
- To assess the impact of storage, DNA purification, and sequencing techniques on bacterial community analysis.
- To provide recommendations for standardized protocols in gut microbiome research.
Main Methods:
- Analysis of fecal samples from 10 individuals.
- Comparison of storage methods: immediate freezing vs. storage on ice.
- Evaluation of DNA purification methods: commercial kits and hot phenol bead beating.
- Assessment of sequencing methods: 454/Roche pyrosequencing (FLX vs. Titanium).
- Comparison of analytical approaches: unweighted (presence-absence) and weighted (abundance) UniFrac.
Main Results:
- DNA purification methods significantly influenced the weighted UniFrac analysis, affecting bacterial abundance data.
- The hot phenol method improved the recovery of Firmicutes sequences.
- Unweighted UniFrac analysis showed less variation due to analytical procedures, with inter-individual variation predominating.
- Storage methods had less impact compared to DNA purification methods.
Conclusions:
- DNA purification is a critical step that can introduce significant variation in gut microbiome studies, particularly when analyzing bacterial abundance.
- For presence-absence based analyses (unweighted UniFrac), variations in analytical procedures have a minimal impact.
- Recommendations are provided for optimizing fecal sample collection, processing, and sequencing to enhance the reliability of gut microbiome research.
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