Phantom mutation hotspots in human mitochondrial DNA

Abstract

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30 000 published hypervariable segment I sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.

Mitochondrial DNA sequence variation in single cells from leukemia patients

“…The percentage of such "novel" mutations was also very high (up to 55%) among the single cells from healthy donors (this study and Ogasawara et al 21 ). Most of these mutations also are not in the list of phantom mutation hot spots that were detected in a massive screening of more than 5000 sequencing electropherograms, 36 and thus are unlikely to be reading errors of sequencing chromatographs. Why these nucleotide changes have only occurred as somatic mutations and are not fixed in human populations as a germline substitution remains an open question.…”

What is a ‘novel’ mtDNA mutation – and does ‘novelty’ really matter?

“…Unusual ensembles of reported mutations may be due to artifacts generated in the course of the electrophoresis Brandstä tter et al 2005). Quite often, such phantom mutations are predominantly transversions (Bandelt et al 2005b;Bandelt and Kivisild 2006).…”

“…It is probably not well known nor greatly appreciated that MITOMAP is a database of rather limited scope and, consequently, quite problematic for two reasons: (1) it actually exhibits and classifies only a non-representative subset of the published mtDNA mutations, and (2) the mutations listed and sorted into different slots are mined from the articles without critical editing; that is, obviously flawed results would also find their way into the database (for a pertinent case, see Brandstä tter et al 2005). The user who innocently takes MITOMAP as the gold standard in the field can thus be seduced into making inadvertent interpretations of his/her mtDNA findings.…”

Mosaics of ancient mitochondrial DNA: positive indicators of nonauthenticity

“…For example, whenever one sees the HVS-II positions 317, 320, 330, 343, and 345 mutated in concert, 39 then the amplified product is a mixture of sequences with elongated C tracts 303 -309 of different lengths. 40 The occurrence of such a phenomenon would also clearly indicate that only the light strand was analysed.…”

“…In a recent screening of 45000 pairs of electropherograms for both strands, site 16095 was among the top four HVS-I sites for which the strands showed discrepant signals due to phantom mutations. 40 It therefore seems that certain sites, such as 16095, may be prone to background noise and phantom mutations under suboptimal sequencing conditions. There are further mutations in the 'Etruscan' data which are otherwise extremely rare (such as the 16098 transition).…”