Erroneous identification of APOBEC3-edited chromosomal DNA in cancer genomics

BACKGROUND: The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5′TpC context. These can be copied as G->A transitions in the 5′GpA context. METHODS: DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA. RESULTS: Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5′GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR. CONCLUSIONS: Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼10(4)–10(5) base substitutions per cancer genome, or 0.003–0.03 kb(−1). By contrast, the 3DPCR error rate is of the order of 4–20 kb(−1) owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5′GpC represent artefacts.