Assessing Deep Sequencing Technology for Human Forensic Mitochondrial DNA Analysis.

摘要

Human mitochondrial DNA (mtDNA) analysis, in a forensic setting, is currently limited in both breadth (the amount of sequence data obtained) and depth (the ability to detect minor variants arising from mutations but present at very low levels). Using emerging technologies, an extension of the breadth of sequence data obtained can easily extend to the entirety of the human mtDNA genome. Extension in the complementary dimension (depth) as reported in this study, has revealed that subtle mixtures are present in forensic DNA samples that are not currently detected by current forensic mtDNA analysis. The ultimate goal of our research effort is to generate whole mt-genome DNA sequence information from limited DNA samples, thus greatly expanding the potential utility of this marker system. We have chosen human hair shaft as a model for these challenging forensic samples. In order to accomplish this goal, we have developed enhanced DNA extraction techniques, performed whole genome amplification of the DNA extracts, employed multiplexed PCR amplification reactions to generate sufficient template mtDNA for NGS applications, and directly sequenced the samples on the Illumina MiSeq instrument. Importantly, we have identified and employed a direct sample preparation step, Nextera, that easily performs DNA library preparation from our enhanced extraction and amplification steps, thus rendering our goal within sight. In this effort, we evaluated two newly emerging methods of DNA sequence analysis to obtain massively parallel mtDNA sequence information (deep sequencing) from hair, buccal, and blood samples. The expanded information available from deep mtDNA sequence analysis revealed that once this new technology is implemented into casework practice, interpretational changes in forensic mtDNA analysis, reflecting the amounts of information that are produced, are necessary. Deep sequencing offers a window into a level of variation that is currently under-appreciated in forensic casework. We have also revealed that the general level of sequence heteroplasmy present in hair shaft samples, as compared to blood and buccal samples, is heightened, but not to a level that would seriously call into question the utility of mtDNA sequencing of hair shaft samples in a forensic context. We demonstrated that careful design of fusion PCR primers supports the creation of amplified targets ready for deep sequencing on both the Roche GS-Junior and Illumina platforms. However, it became clear that this approach was not only unnecessary, but also required much more time and effort than other emerging methods that we explored. We found that using the Nextera-XT kit from Illumina, Inc. allowed us to directly process any double-stranded DNA, including amplicons, for deep sequencing in a much simpler and cost-effective manner. Careful quantitative analysis of amplified DNA molecules allowed us to generate mixtures of DNA templates from different individuals in defined proportions. The deep sequencing results revealed that generally, we could detect a minor variant within a mixture at a 1% level or lower. We noticed that the well-characterized issue of homopolymeric stretches is indeed problematic when performing pyrosequencing reactions. We found that when we directly compared the pyrosequencing results to the results obtained using the Illumina-based chemistry and instrumentation, the ease of identifying the minor variants was significantly enhanced.