Abstract
We assessed the rate of in vitro polymerase errors at polythymidine [poly(T)] tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contain unusually high numbers of poly(T) tracts, and have previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at regions containing more than six consecutive Ts. This artefactual error rate was estimated at 7.3 × 10−5 indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the ‘biological + artefactual’ rate of poly(T) variation. There was no significant difference between the artefactual and the artefactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artefactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred five times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.
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Acknowledgments
Thank you to Ian Riley and Greg Walker for provision of nematode specimens. This work was supported by the Australian Research Council [DP0556520], the University of Wollongong Internationalisation Committee [UIC2007], and the Australian Academy of Science [RI 146].
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Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers HM462006-HM462017.
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Riepsamen, A.H., Gibson, T., Rowe, J. et al. Poly(T) Variation in Heteroderid Nematode Mitochondrial Genomes is Predominantly an Artefact of Amplification. J Mol Evol 72, 182–192 (2011). https://doi.org/10.1007/s00239-010-9414-3
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DOI: https://doi.org/10.1007/s00239-010-9414-3