Investigation and application of methods for ancient DNA research

Abstract

The introduction of high throughput sequencing (HTS) in 2005 caused a revolution in the field of ancient DNA (aDNA). Using the large sequencing capacity of HTS, researchers have overcome the abundant environmental contamination present in most aDNA extractions to reconstruct the genomes of long extinct organisms, such as an archaic horse that perished >500,000 years ago. The proliferation of genomes engendered by HTS has also led to the development of potential ancillary technologies for aDNA research such as genotyping microarrays. In this thesis, HTS and genotyping techniques were developed or refined to improve the application of aDNA to larger biological questions in evolution. This thesis successfully: a) describes an in-house hybridization capture system that uses RNA probes generated from long-range PCR amplicons, b) demonstrates that recombinase polymerase amplification is a less biased alternative to PCR in hybridization capture of aDNA, c) develops an analytical approach that improves phylogenies generated with data from the Illumina BovineSNP50 BeadChip (a commercially available genotyping microarray). In contrast, an attempt to determine the identity of modified nucleotides in aDNA with Pacific Bioscience’s Single Molecule Real-Time (SMRT) sequencing prove to be unsuccessful and genotyping of ancient bison aDNA with the BovineSNP50 BeadChip generated inconsistent results. Furthermore, a hybridization capture probe design was tested and found to be unsuitable for aDNA enrichment. For the larger biological aspect of this thesis, several of the methods developed were used to study bison, because these animals are ideal models of megafauna evolution. Using the in-house hybridization capture system, whole mitochondrial genomes were enriched from aDNA and used to help identify a new extinct species of bison. Furthermore, the new analytical approach for BovineSNP50 BeadChip data was used to demonstrate a significant genetic split between American woods and plains bison, which supports separating these animals at least at the subspecies level. This genetic split suggests that woods and plains bison should be conserved as separate species, which has considerable economic and political implications.