BMC Cancer 2009, 9:113 http://www.biomedcentral.com/1471-2407/9/113 PATIENT 1 PATIENT 2 PATIENT 1 Buccal cells Buccal cells Lymphocytes DNA EXTRACTION DNA EXTRACTION DNA EXTRACTION mtDNA profile mtDNA profile mtDNA profile C16256T C16270T A16220C A16265G T16298C T16362C C16256T C16270T PCR PCR amplicon Dloop1 amplicon Dloop1 PCR amplicon Dloop1 sample mix-up or contamination A16220C T16256C/T A16265G/A T16270C/T T16298C/T T16362C/T TTGE ANALYSIS TTGE ANALYSIS C16256T C16270T COMPARISON OF PROFILES DIFFERENCES INTERPRETED AS SOMATIC MUTATIONS eFDxiigapgularrianemp2ailrlutsotrfatthinegaratseafamcptulealmreixs-uultpsefrvoemnt Tthaant ewtoaul.ld[12] Diagram illustrating a sample mix-up event that would explain part of the artefactual results from Tan et al. [12]. pairs of samples (analyzed with TTGE) there were only five singular changes found other than instabilities of that homopolymeric C tract. In order to minimize the risk of contamination or sample confusion going undetected it is advisable to use at least twice the number (32) of primer pairs for amplification that was employed by Tan et al. [12]. Every nucleotide position should thereby be covered by at least two distinct amplicons, a requirement that e.g. forensic scientists should ideally meet [36]. This would also enhance the chances for TTGE to detect a mutation prior to sequenc- ing. Further note that a good quality electropherogram is no guarantee of an authentic result: when samples are mixed up initially, one can observe perfect sequencing patterns (bands) just showing (seeming) heteroplasmies at those unmatched positions between the two mixed haplotypes. Finally, any discrepancy between the results for two amplicons that indicated a mutated position in one but not the other amplicon would definitely need re- sequencing. In case there are no mutations in the overlap regions of an amplicon with its two neighbouring ampli- cons, then a strict association of the yin-yang pattern with different amplicons gives a strong signal for sample cross- over [29]. Since samples may arrive in the laboratory in problematic state [1], it would be wise to follow some guidelines usually exercised in the field of ancient DNA; in particular, one should perform a critical consideration of all available information and answer the question "Is there any reason to not believe the results or conclu- sions?" [37]. Response By Dr Paul Lewis, Dr Paul Griffiths, Dr Sarah Prior Institute of Life Science, School of Medicine, Swansea University, Swansea, UK In their paper 'Contamination and sample mix-up can best explain some patterns of mtDNA instabilities in buc- cal cells and oral squamous cell carcinoma' Bandelt and Salas have assessed the authenticity of somatic mtDNA mutations observed in oral squamous cell carcinoma (OSCC) reported by Prior et al [11]. The phylogenetic approach used by Bandelt and Salas utilizes publicly avail- able mtDNA sequence data to evaluate whether reported somatic mutations are nothing more than polymor- phisms occurring between different mtDNA haplotypes that co-exist due to sample mix-up. In previous publica- tions Bandelt and Salas have quite rightly called into ques- tion the reporting and authenticity of mtDNA mutations and even suggested 'rules' on error detection and quality control [4,38-40]. We strongly applaud these efforts and thoroughly agree that journals should employ strict rules on provision of sequence data by submitting authors as well as guidelines for journal reviewers. Bandelt and Salas have demonstrated use of their phylo- genetic approach in a previous study of mtDNA muta- tions in different tumour types [4]. In that a posteriori study of sequence data derived from many samples they were able to highlight a number of mutations that were not somatic as originally reported, but simply polymor- phisms between haplotypes occurring in the sample after a likely sample mix-up. We were mindful of the guidelines and problems previously highlighted by Bandelt and Salas Page 5 of 8 (page number not for citation purposes)