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Philos Trans R Soc Lond B Biol Sci. 2015 Jan 19;370(1660):20130624. doi: 10.1098/rstb.2013.0624.

Partial uracil-DNA-glycosylase treatment for screening of ancient DNA.

Author information

1
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA nrohland@genetics.med.harvard.edu.
2
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA Howard Hughes Medical Institute, Boston, MA 02115, USA.
3
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Abstract

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.

KEYWORDS:

ancient DNA; authenticity; barcodes; flexibility; library preparation; target capture

PMID:
25487342
PMCID:
PMC4275898
DOI:
10.1098/rstb.2013.0624
[Indexed for MEDLINE]
Free PMC Article

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