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Researchers Extract and Sequence Ancient DNA From Biofilm Coating Teeth

NEW YORK (GenomeWeb) – Researchers have found a method to sequence mitochondrial DNA from ancient, skeletal remains without damaging them by sampling a biofilm called "dental calculus" that consists primarily of microbes, but also contains human DNA.

The team — which included researchers from the University of Oklahoma, Arizona State University, Pennsylvania State University, and the University of Zurich — described the technique this week in the American Journal of Physical Anthropology.

The goal was to analyze DNA from six individuals in a 700-year old cemetery in Illinois, but in a way that they did not destroy the remains due to communities' "cultural and ethical concerns, especially regarding destructive analyses," the authors wrote.

While such nondestructive methods exist, most "focus on the extraction of DNA from the surfaces of bones teeth, and skin, which can be problematic if DNA preservation is poor or if surface contamination is high," the researchers added. However, they further said that so-called dental calculus is a "nearly ubiquitous and long-term reservoir of ancient host-associated biomolecules, including human DNA."

Thus, they decided to test whether genome-wide information could be extracted from the DNA in ancient dental calculus. They also analyzed DNA extracted from dentine — the hard tissue of the tooth just below the enamel — of two of the individuals, analyzed DNA extracted from dental calculus of two modern humans for comparison.

They then used in-solution capture technology to enrich for the mitochondrial DNA and performed sequencing on the Illumina MiSeq to an average of seven- to 34-fold per sample. For some samples, they also performed shotgun sequencing.

The researchers found that the amount of human DNA found in the mostly microbial biofilm varied greatly. However, they were able to extract more total DNA from the dental calculus than the dentine by an average of more than 25-fold.

Further, in the two modern samples, human DNA consisted of 9.1 percent and 0.1 percent of the total DNA, with mtDNA accounting for only 0.002 percent of the total for both samples. Similarly, the archaeological samples also contained low proportions of mtDNA — on average around 0.0001 percent to 0.0002 percent. But after target capture and enrichment, the proportion of mtDNA increased to 0.41 percent and 1.37 percent in the dental calculus and dentine, respectively.

As expected, the DNA from both dental calculus and dentine was highly fragmented and also damaged. The damage patterns were "consistent with ancient DNA and support the authenticity of the human reads in our data sets," the authors wrote.

They were able to reconstruct the full mitochondrial genome from all samples, and assign mitochondrial haplogroups to each one, including for three samples from which researchers were previously unable to obtain mtDNA via conventional PCR techniques. This highlighted "the importance of newer ancient DNA extraction and next-generation sequencing methods in successfully recovering genetic material from ancient specimens," the authors wrote.

All six archaeological samples could be traced back to Native American ancestry, as expected.  Comparing the mitogenomes of DNA extracted from dentine versus dental calculus, the team noted that they were able to get more mtDNA reads from the dentine, resulting in higher coverage. Thus, while dentine may be a preferable sample, "dental calculus is a suitable substitute," the authors wrote.

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