true-Single Molecule Sequencing Illuminates the Sequencing of Ancient DNA Molecules

Some environmental conditions are compatible with the survival of DNA molecules in fossils over 50,000 years. However, those are generally extremely damaged, by extensive post-mortem hydrolytic reactions that fragment megabase-long chromosomes into short fragments and deaminate thymine residues into uracil residues. Such DNA damages limit our ability to access ancient DNA templates over fresh DNA contaminants that mainly originate from environmental microbes and limit sequencing of ancient individual genomes and extinct species. Third generation sequencing approaches do not depend on complex enzymatic treatments for building sequenceable DNA libraries and are truly amplification-free. Using Helicos true Single-Molecule DNA Sequencing (tSMS) and Pleistocene horse bones preserved in permafrost, we demonstrated that a greater fraction of sequence reads could be mapped back to the horse reference genome, as compared to Illumina reads, confirming that the molecular tools used for Illumina libraries were refractory to a fraction of endogenous DNA molecules. We tested the performance of modifications to the standard Helicos procedure, including the usage of milder denaturation temperature, phosphastase treatment, and oligonucleotide spiking and showed that these could greatly increase the amount of sequence information recovered. We identified molecular preservation niches in bone crystals. Altogether, these procedures could be used to increase sequence coverage of ancient genomes. Finally, tSMS identified for the first time the presence of 3'-overhangs in ancient DNA, a feature that has recently been confirmed with single-strand based Illumina DNA libraries. Our work illustrates how next-generation-sequencing platforms could be turned into high-sensitivity instruments revealing biochemical features of ancient DNA.