16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform
Open accessAbstract
BACKGROUND: Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. RESULTS: The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. CONCLUSIONS: Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
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Almost all cases in which sequencing platforms differed specifically investigated Ion Torrent 16S amplicon sequencing.
“Almost all cases in which platforms differed31–33 specifically investigated Ion Torrent 16S amplicon sequencing”
From Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium (2017) · cites this paper as [33]
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- Authors
- Fouhy, F., Clooney, A. G., Stanton, C., Claesson, M. J., & Cotter, P. D.
- Affiliations
- Teagasc - The Irish Agriculture and Food Development Authority (IE)
- University College Cork (IE)
- Corresponding author
- Claesson, Marcus J.
- Cotter, Paul D.
- Venue
- BMC Microbiology
- Published
- 2016-06-24
- Volume
- 16
- Issue
- 1
- Pages
- 123-123
- ISSN
- 1471-2180 (ISSN-L)
- PMC
- PMC4921037
- OpenAlex
- W2464743969
- Cited by
- 310
- References
- 32
- Citations by year
- 2026: 17 · 2025: 30 · 2024: 15 · 2023: 29 · 2022: 24 · 2021: 51 · 2020: 45 · 2019: 35 · 2018: 36 · 2017: 24 · 2016: 4
- FWCI
- 17.15
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- BMC Microbiology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Arrow@dit (Dublin Institute of Technology) (acceptedVersion)
- Cork Open Research Archive (University College Cork, Ireland) (submittedVersion, cc-by)
- T-Stór (Teagasc) (submittedVersion, cc-by-nc-sa)
- Funders
- Science Foundation of Ireland (SFI/12/RC/2273, 11/PI/1137)
- European Commission (603038)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Keywords
- 16S rRNA gene sequencing, mock microbial community, DNA extraction methods, sequencing platform comparison, MiSeq, bacterial community composition
- MeSH terms
- Bacteria, DNA, Bacterial, RNA, Ribosomal, 16S, DNA Primers, High-Throughput Nucleotide Sequencing, Microbiota
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