16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice
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BACKGROUND: Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning. RESULTS: The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised "universal" PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers. CONCLUSIONS: This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.
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A modified forward primer was used to ensure adequate amplification, including taxa previously underrepresented using conventional primers.
“To ensure adequate amplification, including taxa that were previously underrepresented using conventional primers that target the V1-2 region such as Bifidobacteria [29], a modified forward primer was used”
From Uncovering the trimethylamine-producing bacteria of the human gut microbiota (2017) · cites this paper as [29]
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- Authors
- Walker, A. W., Martin, J. C., Scott, P., Parkhill, J., Flint, H. J., & Scott, K. P.
- Affiliations
- University of Aberdeen (GB)
- Wellcome Sanger Institute (GB)
- Corresponding author
- Scott, Karen P.
- Venue
- Microbiome
- Published
- 2015-06-22
- Volume
- 3
- Issue
- 1
- Pages
- 26-26
- ISSN
- 2049-2618 (ISSN-L)
- PMC
- PMC4482049
- OpenAlex
- W1679625911
- Cited by
- 273
- References
- 59
- Citations by year
- 2026: 20 · 2025: 16 · 2024: 13 · 2023: 20 · 2022: 25 · 2021: 32 · 2020: 26 · 2019: 27 · 2018: 30 · 2017: 32 · 2016: 29 · 2015: 3
- FWCI
- 13.61
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Microbiome (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Aberdeen University Research Archive (Aberdeen University) (submittedVersion)
- Funders
- Wellcome Trust (grant number 098051, 098051)
- Rural and Environment Science and Analytical Services Division
- Scottish Government
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Health Sciences › Nursing › Nutrition and Dietetics › Infant Nutrition and Health
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Keywords
- infant gut microbiota, 16S rRNA gene sequencing, primer design, DNA extraction methods, bead beating, Bifidobacterium, breastfeeding, fecal samples, method comparison
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