Identifying NUMT contamination in mtDNA analyses

Abstract

NUMTs are insertions of mitochondrial DNA (mtDNA) sequences into the nuclear genome. They present different degrees of homology and may be present in various copies throughout the genome. Their presence has been identified firstly in the cat genome, and since then in many other species, namely in humans. When highly homologous to mtDNA and/or present in a high number of copies, they may be co-amplified with or instead of the desired mtDNA sequence, thus becoming a source of contamination in mtDNA analyses. This problem varies from species to species, and has been much discussed for human sequence analyses. Since pets have been more and more targeted in forensics, it is important to understand the extent to which NUMT sequences may interfere with the results of mtDNA analysis in these species. The domestic cat represents a particular case due to the high prevalence of NUMTs. We have performed amplification and sequencing of the cat mtDNA control region ( n ≈ 30) and of ND5 and ND6 genes ( n ≈ 70) using newly designed and previously described primers, respectively. We analysed the sequences with different softwares and constructed phylogenetic networks. By analysing and comparing phylogenies resultant from both sequenced regions we concluded that amplification with control region primers resulted in NUMT contamination. We here discuss how it is possible to distinguish the two kinds of sequences through a careful observation of electropherograms, alignments and phylogenetic networks and recommend critical analyses to be performed after obtaining the sequences in order to safely assign sequence origin.