2019 Winner: Enrichment of Damaged DNA by Target Replacement of Deaminated Cytosines

Project Information
Enrichment of Damaged DNA by Target Replacement of Deaminated Cytosines
Physical and Biological Sciences
UCSC Paleogenomics Lab
Explosive development of next generation sequencing technology has allowed for more extensive use of degraded samples in genomic analysis. Ancient DNA (aDNA) is extracted from samples several hundred to tens of thousands of years old and is often heavily fragmented, contaminated with modern DNA, and contains damaged bases. The most common form of detectable DNA damage is the deamination of cytosines to uracils and is used as an authentication method for aDNA. We aim to exploit this common form of aDNA damage by enzymatically replacing uracil bases with biotinylated nucleotides which allows for the target enrichment of authentic ancient DNA from modern contamination. After creating a single-stranded library and synthesizing a second strand, we enzymatically excise uracils and fill in the gap with a biotinylated nucleotides using a strand-displacing polymerase. With this biotin incorporation, we can pull down molecules with DNA damage onto a streptavidin bead, then PCR-amplify the molecules. We anticipate that the use of our protocol will increase the proportion of informative content in a DNA sequencing library. Libraries with a higher proportion of informative molecules increases the sequencing power of a single library. We hope this technique will provide a tool for researchers to better study the most poorly preserved specimens. With improved access to rare genomic data, we obtain a snapshot into the natural history of population structures and seek to understand current population dynamics in a quickly changing climate.
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Students
  • Lourdes Marcela Gomez (Crown)
Mentors