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Partial uracil–DNA–glycosylase treatment for screening of ancient DNA
Open accessAbstract
The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.
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“We treated the libraries with Uracil–DNA glycosylase (UDG) to reduce characteristic ancient DNA damage at all but the first and last nucleotides29”
From Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation (2018) · cites this paper as [29]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“We assessed the quality of each library at the screening stage using three standard methods for determining ancient DNA authenticity.”
From Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation (2018) · cites this paper as [29]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Large-scale projects can benefit from nascent automation procedures both for the preparation of DNA libraries and their capture 49,126,144 .”
From Ancient DNA analysis (2021) · cites this paper as [126]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“We evaluated each individual for evidence of authentic ancient DNA by limiting to libraries with a minimum of 3% cytosine-to-thymine errors at the final nucleotide29, by requiring that the ratio of X-to-Y-chromosome sequences was characteristic of either a male or a female, by requiring >95% matching to the consensus sequence of mitochondrial DNA30, and by requiring (for males) a lack of variation”
From Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation (2018) · cites this paper as [29]
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“Performing two successive rounds of enrichment generally increases on-target coverage rates 166 , while the use of short adapters can favor on-target library-to-probe annealing and enhance enrichment efficacy 126 .”
From Ancient DNA analysis (2021) · cites this paper as [126]
In the citing paper
“To reduce potential errors in the obtained DNA sequences, we partially removed DNA damage resulting from cytosine deamination using uracil-DNA glycosylase and endonuclease VIII as previously reported (31). In this procedure, the damage is retained in the terminal positions of the DNA fragments and can be leveraged as a measure of aDNA authentication.”
From Ancient DNA sheds light on the genetic origins of early Iron Age Philistines (2019) · cites this paper as [31]
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Metadata
- Authors
- Rohland, N., Harney, E., Mallick, S., Nordenfelt, S., & Reich, D.
- Affiliations
- Broad Institute (US)
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
- Corresponding author
- Rohland, Nadin
- Venue
- Philosophical Transactions of the Royal Society B: Biological Sciences
- Publisher
- The Royal Society
- Published
- 2015-01-19
- Volume
- 370
- Issue
- 1660
- Pages
- 20130624
- ISSN
- 0962-8436 (ISSN-L)
- 1471-2970
- PMC
- PMC4275898
- OpenAlex
- W2154930815
- Cited by
- 572
- References
- 41
- Citations by year
- 2026: 69 · 2025: 64 · 2024: 59 · 2023: 52 · 2022: 59 · 2021: 65 · 2020: 70 · 2019: 46 · 2018: 43 · 2017: 24 · 2016: 15 · 2015: 5
- FWCI
- 9.85
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Philosophical Transactions of the Royal Society B Biological Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Howard Hughes Medical Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Social Sciences › Arts and Humanities › Archeology (arts and humanities) › Paleopathology and ancient diseases
- Keywords
- ancient DNA, library preparation, sample screening, barcoding, cross-contamination, ancient human remains, population genetics
- MeSH terms
- Base Sequence, DNA, Fossils, Genetic Testing, Genetics, Population, Humans, Molecular Sequence Data, Gene Library, Sequence Analysis, DNA, History, Ancient, Uracil-DNA Glycosidase, DNA Barcoding, Taxonomic
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