Examining Mitochondrial DNA Heteroplasmy in Human Hair Shafts using 454 Life Science Next Generation Sequencing
Open Access
- Author:
- McGinley, Kerry Elise
- Area of Honors:
- Forensic Science
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Dr. Mitchell Mark Holland, Thesis Supervisor
Dr. Mitchell Mark Holland, Thesis Honors Advisor
Ronald D Porter, Faculty Reader - Keywords:
- forensic science
mitochondrial DNA
heteroplasmy
hair shafts
454 sequencing - Abstract:
- Through the use of high resolution pyrosequencing the mitochondrial DNA sequence of hair shafts were analyzed for the existence of heteroplasmy. The primer set regions used in forensic mtDNA testing, two and three, were analyzed for five individuals using the 454 GS Junior instrument. Previous studies in our laboratory have shown the reliability of the GS Junior to detect heteroplasmy. This current study focuses on one of the most popular samples, hair shafts that forensic laboratories perform mtDNA testing on. The hair shafts also show the highest rate of heteroplasmy among all other common forensic sample types. The primer set regions were amplified using polymerase chain reaction (PCR) and the primers used to amplify the mtDNA had multiplex identifiers (MIDs) for sample identification and adapter sequences for the pyrosequencing process. After sequencing, the sequence data were analyzed using NextGENe software. The Next Generation ‘primer set 3’ primers used with the MID sequences did not amplify the hair shaft extract DNA due to the unforeseen existence of stable secondary structures of the primers. The Next Generation ‘primer set 2’ primers successfully amplified and sequenced the hair shaft extract DNA. However, there were problems with getting an appropriate coverage or number of reads to examine low-level heteroplasmy. Contamination was an initial concern because of the increased sensitivity of Next Generation sequencing, however results showed minimal contamination and the correct mtDNA profile was generated using the 454 GS Junior to perform the sequencing.