As submitted by the proposer: Human mitochondrial DNA 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, generally referred to as Next-Generation DNA Sequencing (NGS) an extension of the breadth of sequence data obtained from a sample can be expanded to encompass the entirety of the human mtDNA genome. Extension in the complementary dimension (depth) is also possible using NGS and will reveal subtle mixtures that are currently not detected by existing protocols. NGS technologies have the promise of providing information in both of these dimensions, thereby expanding the utility of mtDNA analysis in forensic science. Challenging forensic DNA samples extracted from, for instance, bones and hair can be degraded and/or contain very little DNA. Mitochondrial DNA (mtDNA) analysis is often utilized on these kinds of samples. Studies employing newly emerging DNA sequencing technologies have been designed to interrogate targets down to the single molecule level. Such studies have shown tissue differences (heteroplasmy) within individuals that have lead to recent public calls for a re-evaluation of current interpretational approaches to forensic mtDNA comparisons. We have performed mixture studies on human DNA templates and shown that it is possible to detect minor variants at the 1% level using these new chemistries and instruments. In the proposed effort, we will continue to evaluate newly emerging methods of DNA sequence analysis on the Illumina MiSeq to obtain massively parallel mtDNA sequence information (deep sequencing) from hair shaft, buccal, bone, and blood samples. The expanded information available from deep mtDNA sequence analysis will reveal whether or not interpretational changes in forensic mtDNA analysis of such samples are necessary, as well as the extent to which deep sequencing can offer a window into a level of variation that is currently not attainable in forensic casework. As an ancillary benefit, our efforts will continue to reveal the general level of sequence heteroplasmy present in hair and bone samples as compared to blood and buccal samples, all common targets of forensic mtDNA analysis. Whole genome amplification (WGA) is a promising new approach to pre-amplify samples to levels that can then support downstream operations. These methods employ all the necessary components for DNA synthesis, and attempt to copy the DNA extracted from an evidentiary sample so that sufficient DNA then exists for either targeted PCR or non-specific processing steps that prepare the DNA for next-generation sequencing. We have generated preliminary data that suggests that a streamlined analysis process potentially including a WGA pre-amplification step can be applied to forensic DNA samples. We propose to further evaluate the pre-amplification step in light of the entire workflow in order to quantitatively identify the circumstances wherein the WGA step may be necessary and those instances where it may not be necessary. We will further evaluate a multiplexed PCR amplification strategy using primer sets designed around the human mt-genome. Our approach prepares the samples for direct sequencing on the MiSeq instrument from Illumina, Inc. This process employs a commercially available tagmentation reaction, called Nextera-XT - that enzymatically prepares the DNA for NGS. Our extensive experience with this library preparation method has determined that it is robust, simple to use, results in reproducible sequence data from the same samples, and can enable multiplexing using a series of barcode sequences that can be bioinformatically sorted out at the conclusion of the run. This process will be evaluated for both DNA-limited samples such as hair shafts and bones, as well as known samples.