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Monday, October 28, 2013

Ancient DNA from prehistoric Bulgaria and Denmark

A paper at the AJHG describes a new cost effective method of significantly increasing the amount of authentic DNA output from ancient samples:

By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold.

This is particularly good news for studies which aim to extract autosomal DNA from hundreds of ancient remains, like Gothenburg University's The Rise project, which I excitedly blogged about earlier this year (see here). In fact, I suspect the aforementioned Danish hair sample is one of the samples from The Rise dataset. The reason I say that is because Morten Allentoft is a co-author on this paper, and he's also doing the DNA analysis for The Rise (see here).

In any case, below are two global Principal Component Analyses (PCA) featuring one of the ancient Bulgarians (V2) and the ancient Dane (M4). The principal components (PC1 & 2) were computed using only modern samples, and then the ancient samples projected onto the PCA space.

The ancient Bulgarian is sitting more or less where modern Bulgarians are usually found on such global plots. On the other hand, the ancient Dane is clearly shifted towards East Asia and the Americas, and as a result clusters with Finns, which I suppose is somewhat unexpected because that never happens with modern Danes. So either there's a problem with the analysis, like, say, projection bias (see below for more details), or this Bronze Age Dane was in fact more eastern in terms of global genetic affinities than modern Danes. The latter might well be true if, for instance, he was a recent descendant of migrants from the east (like present-day Russia), and/or he harbored more Mesolithic hunter-gatherer ancestry than Danes do today.

Now, here are a couple of PCA limited to European samples from the supplemental data PDF, including another ancient Bulgarian (K8) and the same ancient Dane (M4). Unfortunately, PC1 appears to be mostly a reflection of the well documented and very recent founder effect and strong genetic drift experienced by the Finnish population. In other words, it's not saying much more than the fact that the ancient samples weren't affected by the same demographic events and genetic drift as Finns during the past few hundred years. It might have been possible to get more informative results by reducing the Finnish sample to only a handful of the least drifted (ie. least Finnish-like) individuals.

Moreover, it's curious that both ancient samples land in more or less the middle of their respective plots in PC2, despite the fact that they come from very different parts of Europe. I suspect that in these instances projection bias is indeed the problem.

Projection bias is similar to the "calculator effect" (see here), but it affects PCA, especially PCA that include only closely related populations, like from Europe. For more background see Haasl et al. 2012 and Lee et al. 2012.

It's also interesting to note that two of the Iron Age Bulgarians are reported as belonging to mtDNA haplogroups U3b and HV, respectively. Both of these haplogroups are generally accepted to be of Near Eastern origin. They're rare in Europe today (usually <2%), but relatively more common in Bulgaria than most other European countries. This suggests some genetic continuity in Bulgaria from at least the Iron Age to the present. Indeed, U3 has been reported from early Neolithic samples from Germany and Ukraine, which means that the ancient Bulgarian U3 lineage need not have arrived in Europe from the Near East during the metal ages.


Carpenter et al., Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries, The American Journal of Human Genetics (2013),

See also...

More info on two Thracian genomes from Iron Age Bulgaria + a complaint

PCA projection bias in ancient DNA studies