Assessing the fidelity of ancient DNA sequences amplified from nuclear genes

Ancient DNA and Evolution Group, Centre for Ancient Genetics, Niels Bohr Institute, University of Copenhagen, Denmark.
Genetics (Impact Factor: 4.87). 02/2006; 172(2):733-41. DOI: 10.1534/genetics.105.049718
Source: PubMed

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.

Download full-text


Available from: Ross Barnett, Jul 28, 2015
  • 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.
    Molecular Ecology Resources 05/2014; 14(3):606-615. DOI:10.1111/1755-0998.12205 · 5.63 Impact Factor
  • 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.
    Science China Earth Science 03/2014; 57(3):388-396. DOI:10.1007/s11430-013-4702-6 · 1.34 Impact Factor
  • Source
    • "For taxonomic assignment, we adopted a conservative approach to avoid incorrect conclusions due to sequencing or PCR error. We assigned only those sequences that differed by a maximum of two nucleotides from those matching from the databases and retained also sequences with type 1 and type 2 transitions substitutions (adenine ↔ guanine and thymine ↔ cytosine) that are typically present in fragments amplified from damaged aDNA templates (Hansen et al. 2001; Binladen et al. 2006). Sequences with more than two nucleotides difference were not assigned and were considered of unknown plant origin, even if they could be identified to family or higher taxonomic levels. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen-based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species-specific primers. The pollen analyses revealed a larger number of taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species-specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigating past flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species-specific primers to provide the most comprehensive signal from the environment.
    Molecular Ecology 04/2013; DOI:10.1111/mec.12298 · 5.84 Impact Factor
Show more