Developing and Applying Molecular Methods for Degraded DNA
Researchers working with poor-quality samples have developed a suite of methods to maximize data generation from minute quantities of short and damaged DNA. Library preparation, the process of preparing extracted DNA for sequencing, is a particularly important step when working with degraded samples. Library preparation approaches optimized for degraded DNA produce more informative libraries but tend to be more laborious, costly, and have lower throughput compared to conventional approaches. In this dissertation, I present a rapid and cost-effective single-stranded DNA library preparation protocol to prepare degraded DNA for Illumina sequencing and apply the approach to several degraded sample types. In the first chapter, I present the first iteration of the library preparation method and demonstrate the effectiveness on cell-free DNA and synthetic oligonucleotides. In the second chapter, I optimize the library preparation method for highly degraded ancient samples and compare the efficacy to two commonly used degraded DNA optimized approaches. In the last chapter, I develop a workflow for whole genome sequencing of single hair shafts and characterize the variation of DNA recovered from the hairs of 50 anonymous volunteers.