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Ancient biomolecules have become an increasingly important part of archaeological investigations interested in understanding population movements and health. Despite their ability to elucidate historically-attested contexts of human mobility and interaction between different cultural groups, biomolecular techniques are still underutilized in certain historical and archaeological contexts. One such context is the Roman Imperial limes, or border zone, along the lower reaches of the Danube, which saw more than five hundred years of migration, conflict, and accommodation among a wide range of populations, from Mediterranean settlers to steppe pastoralists. In this region, more than a century of archaeological investigation has unearthed the remains of tens of thousands of Roman-era individuals. However, only a limited number of contexts have undergone biomolecular analyses. While these deceased humans may offer an untapped reservoir of biomolecular information, many were collected during a period when the standard precautions and protocols for ancient biomolecular research were not yet established. Because contamination is a major barrier for successfully recovering ancient DNA and proteins, conducting a pilot study to assess bimolecular preservation of a small representative dataset of human remains before embarking on a more extensive research program may prevent unnecessary sampling. This study applies ancient DNA and paleoproteomic techniques to human remains from a Roman-period cemetery at Histria, a site located just south of the Danube at the edge of the Roman province of Moesia Inferior. The individuals from whom we sampled dentin and dental calculus were excavated between the 1940s and 1980s and were housed at the Francisc J. Rainer Institute since. Our results suggest that both microbial and human ancient DNA is preserved in the dental calculus and dentin samples. We also successfully recovered sex-specific amelogenin peptides in tooth enamel from three individuals, including a juvenile. In conclusion, our results are encouraging, signifying the feasibility of future aDNA and paleoproteomic research for this skeletal collection. Our analyses also showcase how sex estimation with genomic and proteomic methods may contradict traditional osteological approaches. These findings not only offer deeper insights into the lives of these individuals but also show promise for the investigation of broader anthropological questions, such as the impact of Roman annexation in this region.
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Wet Lab Protocols Matter: Choice of DNA Extraction and Library Preparation Protocols Bias Ancient Oral Microbiome Recovery
ABSTRACT Ancient DNA (aDNA) analysis of archaeological dental calculus has provided a wealth of insights into ancient health, demography and lifestyles. However, the workflow for ancient metagenomics is still evolving, raising concerns about reproducibility. Few systematic investigations have examined how DNA extraction methods and library preparation protocols influence ancient oral microbiome recovery, despite evidence from modern populations suggesting that they do. This leaves a gap in our understanding of how wet‐lab protocols impact aDNA recovery from dental calculus. In this study, we apply two DNA extraction and two library preparation methods in the aDNA field on dental calculus samples from Hungary and Niger. Samples from each context have similar chronological ages, but differences in their levels of aDNA preservation are notable, providing additional insights into how the efficacy of wet‐lab protocols is impacted by sample preservation. Several metrics were employed to assess intra‐ and inter‐sample variability, such as DNA fragment length recovery, GC content, clonality, endogenous content, DNA deamination and microbial composition. Our findings indicate that both DNA extraction and library preparation protocols can considerably impact ancient DNA recovery from archaeological dental calculus. Furthermore, no single protocol consistently outperformed the others across all assessments, and the effectiveness of specific protocol combinations depended on the preservation of the sample. These findings highlight the challenges of meta‐analyses and underscore the need to account for technical variability. Lastly, our study raises the question of whether the field should strive to standardise methods for comparability or optimise protocols based on sample preservation and specific research objectives.
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- Award ID(s):
- 2235545
- PAR ID:
- 10667397
- Publisher / Repository:
- Molecular Ecology Resources
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 25
- Issue:
- 8
- ISSN:
- 1755-098X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1111/1755-0998.70054
