Low-level variant detection in human mitochondrial DNA using the Illumina MiSeq next-generation sequencing (NGS) platform
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Brandon Chase Smith (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Mark Wilson
Abstract: When challenged by difficult biological samples, the forensic analyst is far more likely to
obtain useful data by sequencing the human mitochondrial DNA (mtDNA). Nextgeneration
sequencing (NGS) technologies are currently being evaluated by the Forensic
Science Program at Western Carolina University for their ability to reliably detect lowlevel
variants in mixtures of mtDNA. The sequence profiles for twenty individuals were
obtained by sequencing amplified DNA derived from the mitochondrial hypervariable
(HV) regions using Sanger methods. Two-person mixtures were then constructed by
mixing quantified templates, simulating heteroplasmy at discrete sites and in defined
ratios. Libraries of unmixed samples, artificial mixtures, and instrument controls were
prepared using Illumina® Nextera® XT and deep-sequenced on the Illumina® MiSeq™.
Analysis of NGS data using a novel bioinformatics pipeline indicated that minor variants
could be detected at the 5, 2, 1, and 0.5% levels of detection. Additional experiments
which examined the occurrence of sequence variation in hair tissue demonstrates that a
considerable amount of sequence variation can exist between hairs and other tissues
derived from a single donor.
Low-level variant detection in human mitochondrial DNA using the Illumina MiSeq next-generation sequencing (NGS) platform
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Created on 4/1/2013
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2013
- Keywords
- Forensic science, Illumina(R) MiSeq(TM), low-level mixtures, Minor variant, Mitochondrial DNA, Next-generation sequencing
- Subjects
- Mitochondrial DNA -- Variation -- Data processing
- Human genetics -- Variation -- Data processing
- Nucleotide sequence -- Data processing