Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone (original) (raw)
Related papers
Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum
PLOS ONE, 2017
Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the pros and cons of sampling the different elements.
Isolating the human cochlea to generate bone powder for ancient DNA analysis
The cortical bone that forms the structure of the cochlea, part of the osseous labyrinth of the inner ear, is now one of the most frequently used skeletal elements in analyses of human ancient DNA. However, there is currently no published, standardized method for its sampling. This protocol describes the preparation of bone powder from the cochlea of fragmented skulls in which the petrous pyramid of the temporal bone is accessible. Using a systematic process of bone removal based on distinct anatomical landmarks and the identification of relevant morphological features, a petrous pyramid is cleaned with a sandblaster, and the cochlea is located, isolated, and reduced to a homogeneous bone powder. All steps are carried out in dedicated ancient DNA facilities, thus reducing the introduction of contamination. This protocol requires an understanding of ancient DNA clean-room procedures and basic knowledge of petrous pyramid anatomy. In 50-65 min, it results in bone powder with endogenous DNA yields that can exceed those from teeth and other bones by up to two orders of magnitude. Compared with drilling methods, this method facilitates a more precise targeting of the cochlea, allows the user to visually inspect the cochlea and remove any residual sediment before the generation of bone powder, and confines the damage to the inner ear region and surface of the petrous portion of fragmentary crania.
World Archaeology, 2020
Advances in NGS sequencing technologies, improved laboratory protocols and new bioinformatic workflows have seen huge increases in ancient DNA (aDNA) research on archaeological materials. A large proportion of aDNA work now utilizes the petrous portion of the temporal bone (pars petrosa), which is recognized as an excellent skeletal element for long-term ancient endogenous (host) DNA survival. This has been significant due to the often low endogenous content of other skeletal elements, meaning that large amounts of sequencing are frequently required to obtain sufficient genetic coverage. However, exclusive sampling of the petrous for aDNA analysis introduces a new set of potential biases into our scientific studies – and these issues are yet to be considered by ancient DNA researchers. This paper aims to outline the possible biases of utilizing petrous bones to undertake aDNA analyses and highlight how these complications may potentially be overcome in future research.
Freshly excavated fossil bones are best for amplification of ancient DNA
Proceedings of The National Academy of Sciences, 2007
Despite their enormous potential for phylogeographic studies of past populations, the impact of ancient DNA analyses, most of which are performed with fossil samples from natural history museum collections, has been limited to some extent by the inefficient recovery of ancient genetic material. Here we show that the standard storage conditions and/or treatments of fossil bones in these collections can be detrimental to DNA "survival". Using a quantitative palaeogenetic analysis of 247 herbivore "fossil" bones up to 50,000 years old and originating from 60 different archaeological and palaeontological contexts we demonstrate that freshly excavated and non-treated, unwashed bones contain six times more DNA and yielded twice as many authentic DNA sequences than bones treated with standard procedures. This effect was even more pronounced with bones from one Neolithic site where only freshly excavated bones yielded results. Finally, we compared the DNA content in the fossil bones of one animal, a c. 3,200 year-old aurochs, that were excavated in two separate seasons 57 years apart. Whereas the washed and museum-stored fossil bones did not permit any DNA amplification, all the recently excavated bones yielded authentic aurochs sequences. We established that during the 57 years, during which the aurochs bones were stored in a collection, at least as much amplifiable DNA was lost as during the previous 3,200 years of burial. This result calls for a revision of the post-excavation treatment of fossil bones to better preserve the genetic heritage of past life forms.
American Journal of Physical Anthropology, 2006
We present a method to distinguish authentic ancient DNA from contaminating DNA in a human bone. This is achieved by taking account of the spatial distribution of the various sequence families within the bone and the extent of degradation of the template DNAs, as revealed by the error content of the sequences. To demonstrate the veracity of the method, we handled two ancient human tibiae in order to contaminate them with modern DNA, and then subjected segments of the bones to various decontaminating treatments, including removal of the outer 1–2 mm, before extracting DNA, cloning, and obtaining a total of 107 mitochondrial DNA sequences. Sequences resulting from the deliberate contamination were located exclusively in the outer 1–2 mm of the bones, and only one of these 27 sequences contained an error that could be ascribed to DNA degradation. A second, much smaller set of relatively error-free sequences, which we ascribe to contamination during excavation or curation, was also located exclusively in the outer 1–2 mm. In contrast, a family of 72 sequences, displaying extensive degradation products but identifiable as haplogroup U5a1a, was distributed throughout one of the bones and represents the authentic ancient DNA content of this specimen. Am J Phys Anthropol, 2006. © 2006 Wiley-Liss, Inc.
The suitability of bones and teeth as sources of DNA in ancient bones and teeth
The majority of ancient DNA studies on human specimens have utilised teeth and bone as a source of genetic material. In addition to the abundance of such samples, they are perceived to be a source of uncontaminated endogenous DNA. In this paper the levels of endogenous contamination * (i.e. present within the sample prior to sampling for the DNA analysis) are assessed within a range of European archaeological human bone and teeth samples. A series of experiments are also undertaken to artificially contaminate samples that have previously been assessed for biochemical and histological preservation. The findings demonstrate two important issues: a) although teeth are more resilient to contamination than bone, both are readily contaminated through handling or washing, and b) once contaminated in this way, both are difficult (if not impossible) to decontaminate. The specific biochemical characteristics of individual samples reveal that contamination is directly correlated to sample preservation, and in particular, levels of microbial attack and related increases in sample porosity. The implication is that the vast majority of the known archaeological record has been deeply contaminated. * The impetus behind this work was a series of incomprehensible aDNA results on human bone from Repton, Derbyshire, England. See Biddle and Kjolbye-Biddle, 2001, 45-96. 148
Improving access to endogenous DNA in ancient bones and teeth
2015
Poor DNA preservation is the most limiting factor in ancient genomic research. In the vast majority of ancient bones and teeth, endogenous DNA molecules only represent a minor fraction of the whole DNA extract, rendering traditional shot-gun sequencing approaches cost-ineffective for whole-genome characterization. Based on ancient human bone samples from temperate and tropical environments, we show that an initial EDTA-based enzymatic 'pre-digestion' of powdered bone increases the proportion of endogenous DNA several fold. By performing the pre-digestion step between 30 min and 6 hours on five bones, we identify the optimal pre-digestion time and document an average increase of 2.7 times in the endogenous DNA fraction after 1 hour of pre-digestion. With longer pre-digestion times, the increase is asymptotic while molecular complexity decreases. We repeated the experiment with n=21 and t=15-30', and document a significant increase in endogenous DNA content (one-sided pair...