PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample
Open accessAbstract
The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.
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PCR introduces a bias in 16S tables because some amplicon sequences are amplified more efficiently than others.
“Secondly, PCR introduces a bias in 16S tables, as some amplicon sequences will inevitably be amplified more efficiently than others (Acinas et al., 2005;Schloss et al., 2011) (Fig. 2).”
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- Authors
- Acinas, S. G., Sarma-Rupavtarm, R., Klepac-Ceraj, V., & Polz, M. F.
- Affiliations
- Massachusetts Institute of Technology (US)
- Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
- Corresponding author
- Polz, Martin F.
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2005-12
- Volume
- 71
- Issue
- 12
- Pages
- 8966-8969
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC1317340
- OpenAlex
- W2084443943
- Cited by
- 625
- References
- 0
- Citations by year
- 2026: 12 · 2025: 16 · 2024: 27 · 2023: 17 · 2022: 20 · 2021: 42 · 2020: 40 · 2019: 27 · 2018: 27 · 2017: 34 · 2016: 37 · 2015: 44 · 2014: 42 · 2013: 45 · 2012: 37
- FWCI
- 19.73
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Keywords
- PCR artifacts, 16S rRNA gene, bacterioplankton, clone libraries, sequence bias, sequence clustering, diversity estimation
- MeSH terms
- Animals, Plankton, RNA, Ribosomal, 16S, Reproducibility of Results, Gene Library, Polymerase Chain Reaction, Artifacts, Evolution, Molecular, Taq Polymerase
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