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Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences
A G Speksnijder1, G A Kowalchuk, S De Jong
1Department of Zoology, Natural History Museum, South Kensington, London SW7 5BD, United Kingdom. a.speksnijder@abdn.ac.uk
Abstract:
A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.
Insights
PCR-cloning introduces significant artifactual sequence variation in 16S ribosomal DNA (rDNA) clone libraries. At least 14% of clones showed aberrations due to polymerase errors, mutations, heteroduplexes, and chimeras, explaining microheterogeneity.
Area of Science:
- Microbiology
- Molecular Biology
- Bioinformatics
Background:
- 16S ribosomal DNA (rDNA) sequencing is crucial for microbial community analysis.
- High sequence microheterogeneity in environmental clone libraries often complicates interpretation.
- Sources of artifactual variation in PCR-cloning are not fully understood.
Purpose of the Study:
- To identify and quantify sources of artifactual sequence variation in 16S rDNA clone libraries.
- To assess the impact of PCR-cloning artifacts on microbial community profiling.
Main Methods:
- A defined mixture of seven closely related 16S-rDNA clones was used as a template.
- PCR-cloning experiments were performed to generate clone libraries.
- Recovered clones were sequenced and analyzed for sequence aberrations.
Main Results:
- At least 14% of the recovered 16S rDNA clones contained sequence aberrations.
- Identified artifact sources include polymerase errors, a mutational hot spot, and the cloning of heteroduplexes and chimeras.
- These artifacts contribute to the observed microheterogeneity in environmental clone libraries.
Conclusions:
- PCR-cloning introduces significant artifactual variation into 16S rDNA sequence data.
- Understanding these artifacts is essential for accurate microbial community analysis.
- The identified artifact sources provide a partial explanation for microheterogeneity in environmental clone libraries.