title: Poly(T) variation in heteroderid nematode mitochondrial genomes is predominantly an artefact of amplification.
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.
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. ahr96@uow.edu.au
authors.forename: Angelique H
authors.initials: AH
authors.lastname: Riepsamen
authors.forename: Tracey
authors.initials: T
authors.lastname: Gibson
authors.forename: Janet
authors.initials: J
authors.lastname: Rowe
authors.forename: David J
authors.initials: DJ
authors.lastname: Chitwood
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Subbotin
authors.forename: Mark
authors.initials: M
authors.lastname: Dowton
mesh_terms: D000818:Animals; D016477:Artifacts; D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D030561:Databases, Nucleic Acid; D003576:Electron Transport Complex IV / Q000235:genetics; D014644:Genetic Variation; D049751:Genome, Helminth; D054629:Genome, Mitochondrial; D054643:INDEL Mutation; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011071:Poly T / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014431:Tylenchoidea / Q000235:genetics*
chemical_list: D004272:DNA, Mitochondrial; D011071:Poly T; D003576:Electron Transport Complex IV; D004259:DNA-Directed DNA Polymerase