True single-molecule DNA sequencing of a Pleistocene horse bone – University of Copenhagen

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22 August 2011

True single-molecule DNA sequencing of a Pleistocene horse bone

Ancient DNA research has long been limited to the study of short mitochondrial sequences. Over the last years, however, paleogenetics has entered into the genomic era by delivering the complete genome sequences of extinct species, such as woolly mammoths, ancient humans and archaic hominins.

This major change in scale is due to the advent of Next-Generation Sequencing (NGS) technologies that exhibit massive sequencing capacities, with millions to billions of sequences generated within no more than days.

One common feature to most ancient DNA extracts is to exhibit extremely high levels of environmental bacterial DNA, making shotgun sequencing particularly cost ineffective. Ancient DNA templates also often show substantial levels of damage, which, compared to modern bacterial molecules, might limit their ability to be properly integrated in DNA libraries and get sequenced.

In contrast to previous NGS technologies, so called third-generation sequencing approaches skip the need for library building and truly sequence single DNA molecules as they are, with minimal template preparation procedures. Helicos Corporate Biosciences has released the first commercially available of these platforms, the HeliScope sequencer, that generates over 20 millions of sequences per channel, and could process up to 50 channels per run while exhibiting the lowest error rates across all third generation platforms currently available.

The typically short sequences generated by Helicos true Single Molecule DNA Sequencing (tSMS) is potentially particularly well fitted to ancient DNA molecules, which are generally extremely fragmented. Using different DNA extracts from a horse bone that has been preserved in a permafrost environment over more than 50,000 years, we have shown that Helicos tSMS performs better than current 2nd generation platforms in accessing to ancient DNA templates.

We developed simple modifications to the template preparation procedure, and to the methods used for extracting DNA, and reveal the existence of DNA preservation niches in bone fossils and even further improve our ability to sequence ancient DNA. These results suggest that more DNA molecules than we previously thought are preserved in fossils, and that the latest generation of sequencing platforms will efficiently complement current approaches for characterizing ancient genomes in an unprecedented manner.

This work has been performed in Ludovic Orlando and Eske Willerslev's groups, in close collaboration with Helicos Corporate Biosciences and the Danish National High-throughput DNA Sequencing Centre.