When the first mitochondrial reference genome was published as the Cambridge Reference Sequence in 1981, mitochondrial genomics in its current form was enabled for all investigational purposes.[i] This work was so instrumental to our understanding and utilization of mitochondrial information that Dr. Venter referred to the work as “the first human genome project”[ii]. Forensic genomics has long embraced Sanger sequencing of the mitochondrial DNA (mtDNA) control region to address human identification needs, with many labs across the globe continuing to use those first techniques. Fortunately, massively parallel sequencing (MPS) has not left this important area of investigation behind and provides the benefits of today’s technology to this underserved space: simplicity, speed, sensitivity, scalability.
As part of this regular publication highlight series, I would like to draw your attention to a handful of recent publications demonstrating how the quality of massively parallel sequencing of the mtDNA genome has facilitated deeper analysis to provide better answers for forensic genomics laboratories.
Developments for operational laboratories
- The Scientific Working Group on DNA Analysis Methods (SWGDAM) recently updated the Interpretation Guidelines for Mitochondrial DNA Analysis Methods to incorporate the considerations of MPS technology with mtDNA analysis[iii]. This is a significant step in the standardization of mtDNA analysis to address the challenges and questions that forensics practitioners were encountering as they transitioned to a new technology. It further sets the stage for MPS as the preeminent approach for mtDNA analysis.
- In my opinion, the most consequential publication of 2018 was the release of the alignment-free database search software SAM 2 and its implementation in the EMPOP mtDNA database[iv]. This update takes an enormous leap forward in the curation and normalization of nomenclature for mtDNA reporting.
- A thoughtful description of the implementation plans and process for assessing whole genome mtDNA in casework samples was put forth by the UNT Center for Human Identification (UNTHSC CHI)[v]. Further work is ongoing at this laboratory and they will undoubtedly continue to highlight the considerations as the approach becomes the standard.
- The Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL) published a developmental validation of a whole genome mitochondrial assay using the Nextera XT technology[vi]. The validation and exhaustive analysis are strong templates for understanding the interpretation of MPS data in an operational setting.
- There was continued, detailed work at the Pennsylvania State University describing the heteroplasmy differences observed across collected sample matrices[vii]. MPS technology allowed the team to investigate with a heightened variant sensitivity, giving stronger confidence in their data. They concluded the correlation of heteroplasmy rates between hair and buccal swabs was significant while the correlation between hair and blood cells did not provide a strong correlation.
Investigation of capture assays for highly degraded samples
The forensic genomics community is unique in its regular dealings with the most degraded (<50bp) or damaged mtDNA samples recovered from highly aged or less-than-ideal storage environments. These samples present challenges that require significant labor with specialized chemistry and data analysis pipelines. While these samples represent a small fraction of the total number to be analyzed, the need to evaluate them is no less diminished. MPS, with its long history of development, provides many tools to enable a more successful investigation. Several laboratories have been investigating capture assays in order to handle these degraded samples.
- The AFMES-AFDIL team performed a thorough evaluation of their mitogenome capture assay with positive results, but also a handful of implementation considerations[viii].
- A multi-site team anchored at the Children’s Hospital Oakland Research Institute (CHORI) combined the results from a mtDNA capture assay with a number of targeted nuclear SNPs in order to enhance human identification capabilities for challenging samples[ix].
- The Institute of Legal Medicine at the Medical University of Innsbruck, working with Grupo de Genética de Poblaciones e Identificación, Instituto de Genética, performed a capture assay for the control region with an extensive discussion highlighting the method, bioinformatics, and considerations for formally introducing the approach[x].
The inherent ability of MPS to provide the full sequence of each amplicon sets the stage for sophisticated algorithms to mine this data further. When coupled with haplogroup information, mtDNA NGS data provides a rich space to evaluate mixtures. A few labs have taken the first steps into this new frontier with undoubtedly more research to come as this potential becomes realized.
- The UNT Center for Human Identification along with the Tallinn University of Technology investigated the application of phased MPS data to mixture identification and saw signs of the potential[xi]. They were able to accurately identify the major contributor in all the samples investigated.
- A team of researchers at the Universities of California in Santa Cruz and Davis along with CHORI tackled mtDNA-based mixture analysis with advanced bioinformatics[xii]. I think the tools they developed are ingenious and may form the foundation for the evolution of this space.
The importance of mitochondrial DNA analysis to the forensic community is currently meeting the power of MPS technologies. In that union, MPS proves it can not only address the challenges of yesterday’s approaches, but also serve as the appropriate technology for the daily needs of operational laboratories. If you have a similar passion for mitochondrial analysis, take a look at the ForenSeq™ mtDNA product line which addresses many of the concerns that have hampered adoption of this method to date. It’s an approachable and scalable chemistry solution, leveraging the industry-dominant Illumina sequencing-by-synthesis (SBS) technology with integrated bioinformatics and data analysis software that simplifies interpretation.
[i] Anderson, S., et al. “Sequence and Organization of the Human Mitochondrial Genome.” Nature, vol. 290, no. 5806, 1981, pp. 457–465., doi:10.1038/290457a0.
[ii] Venter, J. Craig. A Life Decoded: My Genome, My Life. Penguin, 2008.
[iii] Scientific Working Group on DNA Analysis Methods (SWGDAM). Interpretation Guidelines for Mitochondrial DNA Analysis by Forensic DNA Testing Laboratories (2019). https://docs.wixstatic.com/ugd/4344b0_f61de6abf3b94c52b28139bff600ae98.pdf
[iv] Huber, Nicole, et al. “Next Generation Database Search Algorithm for Forensic Mitogenome Analyses.” Forensic Science International: Genetics, vol. 37, 2018, pp. 204–214., doi:10.1016/j.fsigen.2018.09.001.
[v] Churchill, Jennifer D., et al. “Working towards Implementation of Whole Genome Mitochondrial DNA Sequencing into Routine Casework.” Forensic Science International: Genetics Supplement Series, vol. 6, 2017, doi:10.1016/j.fsigss.2017.09.167.
[vi] Peck, Michelle A., et al. “Developmental Validation of a Nextera XT Mitogenome Illumina MiSeq Sequencing Method for High-Quality Samples.” Forensic Science International: Genetics, vol. 34, 2018, pp. 25–36., doi:10.1016/j.fsigen.2018.01.004.
[vii] Gallimore, Jamie M., et al. “Assessing Heteroplasmic Variant Drift in the MtDNA Control Region of Human Hairs Using an MPS Approach.” Forensic Science International: Genetics, vol. 32, 2018, pp. 7–17., doi:10.1016/j.fsigen.2017.09.013.
[viii] Marshall, Charla, et al. “Performance Evaluation of a Mitogenome Capture and Illumina Sequencing Protocol Using Non-Probative, Case-Type Skeletal Samples: Implications for the Use of a Positive Control in a next-Generation Sequencing Procedure.” Forensic Science International: Genetics, vol. 31, 2017, pp. 198–206., doi:10.1016/j.fsigen.2017.09.001.
[ix] Shih, Shelly, et al. “Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples.” Genes, vol. 9, no. 1, 2018, p. 49., doi:10.3390/genes9010049.
[x] Eduardoff, Mayra, et al. “Optimized MtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised MtDNA of Different Age and Origin.” Genes, vol. 8, no. 10, 2017, p. 237., doi:10.3390/genes8100237.
[xi] Churchill, Jennifer D., et al. “Parsing Apart the Contributors of Mitochondrial DNA Mixtures with Massively Parallel Sequencing Data.” Forensic Science International: Genetics Supplement Series, vol. 6, 2017, doi:10.1016/j.fsigss.2017.09.145.
[xii] Vohr, Samuel H., et al. “A Phylogenetic Approach for Haplotype Analysis of Sequence Data from Complex Mitochondrial Mixtures.” Forensic Science International: Genetics, vol. 30, 2017, pp. 93–105., doi:10.1016/j.fsigen.2017.05.007.