Mitochondrial DNA (mtDNA) is often used for analysis in forensic and mass disaster cases involving small and degraded tissue samples. Tissues can degrade from exposure to various weather and environmental conditions. High temperatures and level of destruction can also cause tissues to fuse together or commingle with other remains, resulting in mixtures that can make analysis and interpretation difficult (Budimlija et. al, 2003). While the quality and level of nDNA is low in these cases, mtDNA has a high copy number, increasing the possibility of obtaining enough DNA from compromised samples for fragment-size analysis of short tandem repeats. In addition, the control region of mtDNA contains highly polymorphic hypervariable regions I/II (HVI/HVII) where heteroplasmic mutations have been observed. It is important to understand the nature of heteroplasmy across various tissue types because the presence of heteroplasmy in mtDNA can affect interpretation and analysis.
The Next-Generation Sequencing (NGS) technology overcomes limitations of Sanger sequencing in forensic mtDNA analysis with its high and massively-parallel throughput, clonal amplification, and pyrosequencing chemistry. Through these features, the 454 GS Junior can separate individual components of a mixture, provide a quantifiable estimate of the ratio of mixture components, and analyze low frequency of heteroplasmy in mtDNA. This research project aims to address these common issues in forensic mtDNA analysis by using a sensitive NGS method to characterize low levels of heteroplasmy in brain, heart, muscle, and blood tissues.
In comparison to Sanger sequencing data on the same tissue samples, data from this research project has detected heteroplasmy occurring at frequencies as low as 1.14%. The average heteroplasmic frequency in the HVII region for all heart, muscle, blood, and brain samples in this study was 9%. In the HVII region, muscle samples exhibited the highest average frequency of heteroplasmy of 13% while blood samples exhibited the lowest frequency of heteroplasmy at 2%. In the HVI region, blood samples exhibited the highest average frequency of heteroplasmy of 4% while brain samples exhibited no heteroplasmy. More somatic mutations than germline mutations were observed in all four tissue types and most heteroplasmic samples exhibited heteroplasmy at only one site. “Hot spots” – locations in the mtDNA hypervariable region in which heteroplasmy occurred the most – were observed at positions 64, 185, and 189 in the HVII region. In addition, a statistically significant relationship was determined between heteroplasmy and age using the Chi-square test for independence. However, regression analysis of the sample set indicates that age is not the only factor determining the occurrence of heteroplasmy in these tissue samples.
These results support the high sensitivity of 454 GS Junior not only to detect low levels of heteroplasmy but also to reveal additional heteroplasmic sites in the HVI/HVII regions. In addition to confirming heteroplasmy previously detected by Sanger sequencing, the more sensitive NGS platform has detected additional heteroplasmic instances that were not previously observed in Sanger sequencing. Furthermore, NGS provides a quantitative assessment of heteroplasmy by establishing frequencies of the two bases that occur at one site. This research will help establish NGS sensitivity thresholds for varying levels of heteroplasmy in different sample types. Moreover, the results demonstrate the potential of NGS to improve interpretation guidelines and increase the efficiency of forensic mtDNA analysis, especially with limited and degraded DNA samples.
(Publisher abstract provided.)
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