Tuesday, 13 November 2012

Paper of the week - Next gen sequencing of ancient DNA

This paper was featured in the 12 October issue of Science, so really isn't from this week, nevertheless I thought it would be relevant given the previous post on library preparation.

Sequencing ancient DNA is a hugely challenging task. Not only is it very difficult to get any sort of yield of DNA from bones tens of thousands of years old, but the DNA itself is normally degraded to such an extent that conventional library preparation is highly inefficient. On top of this, there is the challenge to eliminate environmental contamination. To avoid much of this, the team, lead by Matthias Meyer at the Max Planck Institute for in Leipzig came up with a simple but efficient method to generate sequencing libraries from single stranded DNA. The basic steps in the library prep are:

  • Dephosphorylate
  • Heat denature
  • Ligate single stranded biotinylated adaptors
  • Immobilize on streptavidin beads
  • Generate the second strand with DNA polymerase
  • Ligate a second adaptor by blunt end ligation

Figure 1 from Meyer et al is the new strategy for library preparation.

The main benefit of this prep over standard techniques is the hugely increased yield (over 6 fold more library), as well as the ability to identify the ends of the molecule accurately and analyse strand breakage. The authors suggest that strand breaks occur at a higher frequency near guanines. On the downside to this method, I foresee some bias introduced by the ligation steps. Single stranded ligation of microRNA to adaptors in the small RNA library prep with T4 RNA ligases is known to be a biased process (paper, paper). Nevertheless, at high coverage levels and long reads (150 nt), the bias shouldn't necessarily lead to false conclusions in genome sequencing.

I see this library preparation method having a big impact in a few ways outside of the molecular archaeology field. I think it will make it easier to analyse degraded DNA from diverse types such as formalin-fixed, paraffin-embedded, dried blood spots, forensic samples and environmental DNA (eg, from soil). 

The other reason I think this is big news is that it could be useful in the field of epigenetics. Generating libraries for shotgun bisulfite sequencing requires micrograms of starting material to get just a tiny amount of library because the bisulfite treatment often leaves the DNA in a highly degraded state, consisting mostly of single strands. Moreover, methods like ChIP-Seq often need to get quality libraries from just a few nanogram of DNA, and if methods like this one from Meyer can improve yield then it is a big win. 

In addition to this new library prep method, the paper is well worth a read for its insights into human evolution over the last million years. Enjoy!

[Edit: the very detailed supplementary material is well worth a read!]

Science. 2012 Oct 12;338(6104):222-6. doi: 10.1126/science.1224344. Epub 2012 Aug 30.A high-coverage genome sequence from an archaic Denisovan individual.Meyer M, Kircher M, Gansauge MT, Li H, Racimo F, Mallick S, Schraiber JG, Jay F, Prüfer K, de Filippo C, Sudmant PH, Alkan C, Fu Q, Do R, Rohland N, Tandon A, Siebauer M, Green RE, Bryc K, Briggs AW, Stenzel U, Dabney J, Shendure J, Kitzman J, Hammer MF, Shunkov MV, Derevianko AP, Patterson N, Andrés AM, Eichler EE, Slatkin M, Reich D, Kelso J, Pääbo S.
Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany.
We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of "missing evolution" in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.
PMID: 22936568 [PubMed - indexed for MEDLINE]