Forensic DNA samples are often highly degraded, making them unsuitable for traditional methods of DNA analysis, such as STR analysis, because target sequences and primer binding sites are not intact. Mitochondrial DNA (mtDNA) is useful for analyzing degraded DNA because of its high copy number; the high copy number makes successful typing more likely. Traditional methods of mtDNA analysis, including Sanger sequencing, are commonly used to only target only the HVI/HVII regions of the mitochondrial genome. These analysis methods miss discriminating information outside of the HV1/HVII, limiting the discriminatory power of mitochondrial DNA analysis. Probe capture is a novel technique that uses mtDNA sequence-specific probes to enrich and capture the entire mitochondrial genome. Analyzing the entire mitochondrial genome allows detection of discriminating information outside of the HVI/HVII regions and increases the discriminatory power of mitochondrial DNA analysis. Next Generation Sequencing, a massively parallel, clonal, and high-throughput technique, is an excellent tool for analyzing the entire mitochondrial genome.
The purpose of this project was to test the efficiency of Dr. Calloway’s optimized probe capture assay on degraded DNA by analyzing the entire mitochondrial genome of highly degraded bone samples. Throughout the course of this project, three sets of bones samples, dating to 100, 2000, and 4000 years old respectively, were tested.
Seven highly degraded bone samples recovered from a comingled tomb in Rijeka, Croatia and dating to approximately 100 years old were successfully sequenced with coverage of the mitochondrial genome ranging from approximately 52 to 98%. Haplogroups were determined for six of these samples based on the sequenced variants present in the entire mitochondrial genome. Additionally, six prehistoric bone samples dating to approximately 2000 years ago were sequenced with coverage of the mitochondrial genome ranging from 26 to 100%. The last set of samples, dating to 4000 years old, was recovered from a necropolis on the island of Kor?ula, Croatia. These six samples were successfully sequenced with coverage of the mitochondrial genome ranging from 26 to 100%.
(Publisher abstract provided.)