Next-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. Because of their generally short and degraded/damaged nature, and impurities from modern DNA samples, ancient DNA samples are particularly sensitive to sample preparation biases. Library construction and amplification steps may result in sequences that do not reflect the original DNA template composition. The ligation- and amplification-free nature of True Single Molecule Sequencing (tSMSTM) provides the most unbiased view of the molecular complexity of ancient DNA extracts.
The application of tSMSTMto ancient DNA sample sequencing has been described in the following publication:
True single-molecule DNA sequencing of a Pleistocene horse bone. Genome Res. 2011 Jul 29. [Epub ahead of print]
The Approach
DNA obtained from archival/ancient specimens is modified with a poly-A tail and loaded onto the instrument. No fragmentation, ligation or PCR amplification steps are required. The tailed fragments hybridize to complementary poly-T strands anchored to the flow cell surface. Inside the HeliScope™ Single Molecule Sequencer, a series of nucleotide addition and detection cycles determine the sequence of each fragment. While the sequence data obtained will typically contain both ancient and contaminating modern DNA sequences, alignment of the sequence reads to references which most closely interrogate the ancient DNA allows you to preferentially determine the sequence of the ancient DNA sample. Helicos provides Open source data analysis software designed to align sequence reads to your chosen reference sequence.
Helicos DNA Sample Preparation Methodology