Assessing the fidelity of ancient DNA sequences amplified from nuclear genes

Abstract

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine --> guanine and thymine --> cytosine) and type 2 transitions (cytosine --> thymine and guanine --> adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences.

Figure 1.

Total sequence heterogeneity observed in the mtDNA and nuDNA clone sequences from the 23 specimens (see Table 1). Solid bars, mtDNA total sequence heterogeneity. Shaded bars, nuDNA total sequence heterogeneity.

Figure 2.

Correlation analyses. Colors indicate the preservation environment: blue, permafrost; red, cave and swamp; and green, museum samples. + denotes mtDNA sequences and ○ denotes nuDNA sequences. (A) Type 1 transitions (TS1) as a function of TSH. (B) Type 2 transitions (TS2) as a function of TSH. (C) TS1 as a function of TS2. (D) TS2/TS1 as a function of TSH.