 mtDNA of this Neandertal carries 133 such diagnostic positions (23). The no repair dataset yielded 139 mtDNA fragments that overlapped such positions; 138 carried the Neandertal base while one matched modern human mtDNA. The UDG/endoVIII treated dataset yielded 128 informative fragments, of which all were the Neandertal type. Thus, the mtDNA in all libraries was almost completely free of contamination by modern human mtDNA, even after treatment with UDG and endoVIII. Since the ratio of mitochondrial to nuclear DNA may di?er between the contaminating and the Neandertal DNA, this estimate is strictly applicable only to the mtDNA (33). However, the estimate of mtDNA contamination in these libraries is low enough that within even a few-fold variation in mtDNA:nuclear DNA ratios between the Neandertal and contaminating DNA, sequences aligning to the human nuclear genome will be predominantly of Neandertal origin (Briggs et al. 2009: 9).
A recurring theme in the paper is that very high numbers of resequencing will be necessary to improve the accuracy of ancient DNA. Probing for a particular genetic variant – such as the “Neandertal diagnostic” mtDNA sites – is less of a problem, because there is a low probability of a sequencing error at any particular site. But summed across thousands or millions of base pairs, the number of sequence errors can easily exceed the number of genuine differences between Neandertal and human genomes. For example:
Figure 6 shows that for mitochondrial sequences, overall error rate per base (Figure 6) was 2.20% for no repair sequences; 0.40% for UDG/endoVIII sequences and 0.09% for multi-pass UDG/endoVIII-treated sequences. Thus, UDG/endoVIII treatment alone results in a 5.5-fold reduction in error rates while deep sequencing results in an additional 4.4-fold reduction. In combination this results in a 22-fold reduction in errors. In nuclear DNA, for which we removed CpG sites from the analysis due to the e?ect of methylation described above, a similar pattern is observed (Figure 6) although the true error rate at these low levels cannot be accurately calculated due to genuine sequence di?erences between this Neandertal and the human reference.
A truly impressive low error rate in the best case – which here involves sequencing many copies of the same sites, generating “deep coverage”. But consider: the average sequence divergence between two humans is around 0.1 percent. With a total error rate of 0.09 percent, there would be nearly as many erroneous differences as real ones between an ancient genome and the modern reference. And sequencing two ancient genomes would double the errors, so that the apparent genetic differences would be roughly three times the actual value.
Even deeper coverage will be necessary, hopefully reducing error rates further. This may lead to a hypothesis-testing approach for sequence differences found in Neandertal genomes, as the false positives are sifted out of the data. It’s going to take some creative population genetics to deal with this problem, as we try to analyze the data from these ancient specimens.
Briggs AW, Stenzel U, Meyer M, Krause J, Kircher M, Pääbo S. 2009. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA. Nucleic Acids Res (advance) doi:10.1093/nar/gkp1163