Assessing the Fidelity of Ancient DNA Sequences Amplified From Nuclear Genes

Aarhus University, Aarhus, Central Jutland, Denmark
Genetics (Impact Factor: 5.96). 02/2006; 172(2):733-41. DOI: 10.1534/genetics.105.049718
Source: PubMed


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.

Download full-text


Available from: Ross Barnett
  • Source
    • "The disparity between mtDNA and nuDNA mean fragment length is in agreement with recent studies of vertebrates from permafrost and sediment deposits (Schwarz et al. 2009; Allentoft et al. 2012), which suggest that nuDNA degrades at a faster rate than mtDNA. This may be due to the circular configuration of mtDNA making it less accessible to exonucleases (Allentoft et al. 2012), the double membrane of the mitochondrion offering additional protection (Schwarz et al. 2009) or the interaction of nuDNA and histones facilitating strand breaks (Binladen et al. 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry-stored museum and ancient permafrost-preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterisation of extracted DNA demonstrated that parasite-derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect. This article is protected by copyright. All rights reserved.
    Full-text · Article · May 2014 · Molecular Ecology Resources
  • Source
    • "Both as typical members of the " Coelodonta-Mammuthus Fauna " in late Pleistocene, the woolly rhinoceros and the woolly mammoth have received unequal attentions at the molecular level. The phylogenetic position of the woolly mammoth has been fully resolved by multiple sampling locations (Yang et al., 1996; Noro et al., 1998; Lister et al., 2001; Debruyne et al., 2003; Krause et al., 2006; Rogaev et al., 2006; Yang et al., 2006; Gilbert et al., 2008; Miller et al., 2008; Enk et al., 2009), whereas DNA studies on Coelodonta antiquitatis were focused only on very limited sampling sites in Europe and northern Asia (Orlando et al., 2003; Binladen et al., 2006; Willerslev et al., 2009; Lorenzen et al., 2011). Among the interested issues of the woolly rhinoceros, the close phylogenetic relationship between this species and the modern Sumatran rhinoceros was accepted by previous ancient DNA studies (Orlando et al., 2003; Willerslev et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ancient DNA data have supported a sister relationship between woolly rhinoceros and extant Sumatran rhinoceros. This relationship has been used to explore the divergent times for the woolly rhinoceros from their relatives. Complete and partial ancient DNA sequences of the mitochondrial cytochrome b (cyt b) gene were retrieved from bones of the late Pleistocene Coelodonta antiquitatis excavated from northern and northeastern China. The newly obtained sequences together with the European and northern Asian Coelodonta antiquitatis sequences from GenBank were used to estimate the evolutionary divergence time. Phylogenetic analyses showed the exchange of genetic information between the Chinese individuals and Coelodonta antiquitatis of north Asia, which also indicated a more recent evolutionary timescale (3.8–4.7 Ma) than previous molecular estimations (17.5–22.8 or 21–26 Ma) for woolly rhinoceros based on the fossil calibration of outgroups. This new timescale was more consistent with the fossil record of the earliest known genus Coelodonta.
    Full-text · Article · Mar 2014 · Science China Earth Science
  • Source
    • "This supports the notion that post-mortem miscoding lesions are a negligible source of error in historical specimens [20], [25]. Although we used organellar DNA to assess damage, the sequence error rates in organelle and nuclear genomes have been observed not to differ [32] and, therefore, we expect that our organellar genomic results should be representative of those for nuclear genomes. Whilst the observed sequencing errors are like random noise and unlikely to produce a phylogenetic signal given sufficient read depth coverage, our data could be used for high-confidence genotyping, and selection of SNP and indel-rich coding regions that will allow for genome-scale genetic diversity and phylogenetic analyses. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.
    Full-text · Article · Jul 2013 · PLoS ONE
Show more