BMC Cancer 2009, 9:113 http://www.biomedcentral.com/1471-2407/9/113 The rationale for an a posteriori analysis is that mitochon- drial genomes evolve along a phylogeny (genealogical tree) and are highly polymorphic. This high polymor- phism is only in part due to a minority of extreme hotspot mutations (such as polyC-tract length polymorphisms) but is mainly generated by blocks of inherited mutations that are not reshuffled by recombination as in the case of the diploid autosomal genome. Since many mutations occur only very few times in parallel along the global mtDNA phylogeny, a combination of several such inher- ited mutations locates a sample in the phylogeny in a unique way, rendering it extremely unlikely that a com- plex mutational pattern could have arisen de novo. This allows the researcher who has the necessary knowledge about natural mtDNA variation to question mtDNA sequencing results. Methods We tab into the standard databases in the field, namely MITOMAP [13] and mtDB [14]. In addition, we perform Google searches of the kind described in [15,16]. This directly leads to entire coding-region haplotypes or con- trol-region haplotypes stored in GenBank and discussed in the Web by commercial genetic ancestry companies or their clients. To put the recorded mutations or haplotypes into phylo- genetic context, knowledge about the continental mtDNA phylogenies is drawn from various publications [17-20]. Mutational hotspots with positional mutation rates well above the rate averaged over the entire molecule are read- ily identified by aggregating the macro-haplogroup trees and counting the recurrent changes at each site; see [21] and Table 4 of [22]. Numbering of mutations and poly- morphisms along the mtDNA genome are referred to the revised Cambridge Reference Sequence, rCRS [23]. Results Oral squamous cell carcinoma Prior et al. [11] extracted mtDNA from 30 paired samples of tumour and non-tumour tissue, which was analyzed for contrasting variation within the two short fragments 4527–4954 and 30–407. These ranges constitute the puta- tively readable parts of the amplicons, excluding the primer locations, although it is not clear from their article whether the entire ranges and both strands were in fact analyzed and well readable from start to end. Note that the analysis of both strands and re-sequencing of the same DNA extracts and amplicons do not prevent artificial recombination due to e.g. sample mix-up; only separate and individual extractions from the same individual could provide the minimum guaranties [4]. The observed paired mtDNA sequences were unfortunately not reported in Prior et al. [11], so that no a posteriori check is possible with respect to the potential completeness of the muta- tion profiles compared to the worldwide database of pub- lished mtDNA sequences. The information that is given in the two tables concerns only the nucleotide differences between the two sequences from each pair of analyzed tis- sues. The first table then provides the contrasting variation for the portion 4527–4954 of the ND2 gene. Assuming that the patients had West Eurasian matrilineal ancestries, we first take a look at the basal part of the corresponding mtDNA phylogeny by focussing on the classified parts as reflected in Figure 1 of Palanichamy et al. [17]. Then only seven mutations within that short fragment highlight hap- logroups that were well described before 2005; see our Figure 1. Alternatively, one can search the mtDB database for variation within this fragment and consider, say, the top twelve most frequently recorded variants. Among those, there are seven that predominantly are of East Asian provenance and thus not relevant here. Then the five remaining frequent variants are A4529T, G4580A, A4769G, A4793G, and A4917G, which are – not inciden- tally – all signifying basal haplogroups in Europe (Figure 1). The fact that these variants are frequently stored in the nVFaiegrwruorwoenw1thinedoWwes4t5E2u7r–a4s9ia5n4 mtDNA phylogeny through the View on the West Eurasian mtDNA phylogeny through the narrow window 4527–4954. Boxed letters designate haplogroups and rCRS (revised Cambridge Refer- ence Sequence) and α-γ are specific lineages. Each bar indi- cates a mutation, which is numbered when it occurred within the window. All mutations are transitions unless suffixed by a letter in case of a transversion (to T or C) or a deletion (d). Nomenclature of haplogroup H2 and H2a is as in [20]. Page 2 of 8 (page number not for citation purposes)