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The development of complete mitochondrial genome (mitogenome) reference data for inclusion in publicly available population databases is currently underway, and the generation of more high-quality mitogenomes will only enhance the statistical power of this forensically useful locus. To characterize mitogenome variation in Sweden, the mitochondrial DNA (mtDNA) reads from the SweGen whole genome sequencing (WGS) dataset were analyzed. To overcome the interference from low-frequency nuclear mtDNA segments (NUMTs), a 10% variant frequency threshold was applied for the analysis. In total, 934 forensic-quality mitogenome haplotypes were characterized. Almost 45% of the SweGen haplotypes belonged to haplogroup H. Nearly all mitogenome haplotypes (99.1%) were assigned to European haplogroups, which was expected based on previous mtDNA studies of the Swedish population. There were signature northern Swedish and Finnish haplogroups observed in the dataset (e.g., U5b1, W1a), consistent with the nuclear DNA analyses of the SweGen data. The complete mitogenome analysis resulted in high haplotype diversity (0.9996) with a random match probability of 0.15%. Overall, the SweGen mitogenomes provide a large mtDNA reference dataset for the Swedish population and also contribute to the effort to estimate global mitogenome haplotype frequencies.

Mitochondrial DNA (mtDNA) analysis plays a specialized role in forensic applications, overcoming certain limitations of autosomal DNA markers. The high copy number and uniparental inheritance pattern of mtDNA are advantageous in cases involving shed hairs and aged skeletal elements, especially decades-old missing persons cases. Though the discriminatory power of mtDNA is limited by common haplotypes, next generation sequencing (NGS) offers feasible access to entire mitochondrial genome (mitogenome) data that can provide increased resolution of common haplotypes to unique sequences. The primary implementation challenge of mitogenome analysis is a lack of forensic-quality reference data, which are required to determine the evidentiary weight of a match. A better understanding of NGS methods and data analysis is also necessary to ensure the generation of reliable mitogenome data. Furthermore, appropriate quality control (QC) measures must be established as analysis can be complicated by nuclear mtDNA segments (NUMTs), misalignment of homopolymer regions, sequencing errors, and other artefacts. Including such false variants in mtDNA haplotypes can lead to erroneous conclusions based on misinterpreted data. This thesis aimed to address the implementation challenges of mitogenome analysis and facilitate the transition to NGS in forensic laboratories. Paper I assessed the feasibility of generating forensic-quality mitogenome data from whole genome sequencing (WGS) data, which are valuable sources of mitogenome haplotypes for population studies. Due to NUMT interference, a 10% variant frequency threshold was necessary to produce haplotypes consistent with high-quality mitogenome datasets. Since length heteroplasmy (LHP) can also complicate mtDNA data analysis, Paper II characterized LHP in data generated on two NGS platforms as well as with Sanger-type sequencing. Different patterns of LHP were observed across sequencing technologies, further supporting current guidelines

Whole-genome sequencing (WGS) data present a readily available resource for mitochondrial genome (mitogenome) haplotypes that can be utilized for genetics research including population studies. However, the reconstruction of the mitogenome is complicated by nuclear mitochondrial DNA (mtDNA) segments (NUMTs) that co-align with the mtDNA sequences and mimic authentic heteroplasmy. Two minimum variant detection thresholds, 5% and 10%, were assessed for the ability to produce authentic mitogenome haplotypes from a previously generated WGS dataset. Variants associated with NUMTs were detected in the mtDNA alignments for 91 of 917 (~8%) Swedish samples when the 5% frequency threshold was applied. The 413 observed NUMT variants were predominantly detected in two regions (nps 12,612–13,105 and 16,390–16,527), which were consistent with previously documented NUMTs. The number of NUMT variants was reduced by ~97% (400) using a 10% frequency threshold. Furthermore, the 5% frequency data were inconsistent with a platinum-quality mitogenome dataset with respect to observed heteroplasmy. These analyses illustrate that a 10% variant detection threshold may be necessary to ensure the generation of reliable mitogenome haplotypes from WGS data resources.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3931 autosomal SNPs for extended kinship analysis, and no clinically relevant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict first to fifth degree relationships with strong statistical support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5000 SNP panel for forensic applications.

Given the enhanced discriminatory power of the mitochondrial DNA (mtDNA) genome (mitogenome) over the commonly sequenced control region (CR) portion, the scientific merit of mitogenome sequencing is generally accepted. However, many laboratories remain beholden to CR sequencing due to privacy policies and legal requirements restricting the use of disease information or coding region (codR) information. In this report, we present an approach to obviate the reporting of sensitive codR data in forensic haplotypes. We consulted the MitoMap database to identify 92 mtDNA codR variants with confirmed pathogenicity. We determined the frequencies of these pathogenic variants in literature-quality and forensic-quality databases to be very low, at 1.2% and 0.36%, respectively. The observed effect of pathogenic variant filtering on random match statistics in 2488 forensic-quality mitogenome haplotypes from four populations was nil. We propose that pathogenic variant filtering should be incorporated into variant calling algorithms for mitogenome haplotype reporting to maximize the discriminatory power of the locus while minimizing the reveal of sensitive genetic information.

The World War II Battle of Tarawa, 1943, was a devastating conflict that resulted in losses of more than 1100 American and 4690 Japanese troops. The United States government aims to identify and repatriate the remains of all missing American service members through the Defense Prisoner of War/Missing in Action (POW/MIA) Accounting Agency (DPAA) and its partners such as the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL). Remains associated with the Battle of Tarawa have been recovered from field excavations conducted by History Flight, a DPAA strategic partner, as well as from the National Memorial Cemetery of the Pacific (NMCP) in Hawaii where unknowns have been disinterred for identification. DNA testing at the AFMES-AFDIL has produced mitochondrial DNA (mtDNA) sequences from 1027 case samples to date. Haplogroup assignments indicate that more than one third (36.2 %) of field-collected samples are likely of Asian maternal ancestry. Therefore the field collections from the Tarawa battlefield comprise the remains of American service members but also those of foreign nationals from Asia. The mtDNA of the NMCP unknowns is similar in ancestry proportion to the family reference sample distribution. The DPAA uses the ancestry information gleaned from mtDNA sequence data in conjunction with anthropological evidence to make foreign national determinations. In this way, mtDNA haplogrouping is used to sort the commingled and fragmentary remains recovered from Tarawa between Americans and foreign nationals, which are then repatriated to their country of origin.

A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets. We developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences. The described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing.

The development of mitochondrial genome (mitogenome) reference data for inclusion in publicly-available population databases is currently underway, and the more high-quality mitogenomes that can be generated will only enhance the statistical power of this forensically-useful locus. In order to promote mitogenome testing in Sweden, the mitochondrial DNA (mtDNA) data from the SweGen whole genome sequencing (WGS) dataset were analysed. To avoid the interference from nuclear mtDNA segments (NUMTs), a 10% frequency threshold was applied for the population analyses. In total, 934 forensic-quality mitogenome haplotypes were produced. Despite the elevated frequency threshold, 31 NUMT variants were observed in 13 lower coverage haplotypes. However, NUMT interference was minimal and localized to two hotspot regions, substantially reducing the analysis burden required at lower frequency thresholds. Almost 45% of the SweGen haplotypes belonged to haplogroup H and nearly all mitogenome haplotypes (99.1%) assigned to European haplogroups, which was expected based on previous mtDNA studies of the Swedish population. There were characteristic northern Swedish (i.e., Saami) and Finnish haplogroups observe in the dataset, consistent with the nuclear DNA analyses of the SweGen data. The analysis of the complete mitogenome resulted in high haplotype diversity (0.9996) with a random match probability of 0.15%. Overall, the mitogenomes generated from the SweGen WGS data provide a mitogenome reference database for Sweden as well as contribute to the global effort to increase the availability of mitogenome reference data.

Phylogenetic information can be used to infer maternal biogeographic ancestry and provide a valuable quality control (QC) check of mitochondrial DNA (mtDNA) data, identifying errors in the data such as artificial recombination and phantom mutations. Additionally, the phylogeny guides mtDNA nomenclature, which is crucial for forensic searches. This study compared three haplogrouping tools and their ability to provide proper guidance based on the predicted phylogeny. The Mitochondrial Haplogrouper tool of the AFDIL-QIAGEN mtDNA Expert (AQME), a plug-in for the CLC Genomics Workbench, and the web-based tools HaploGrep2 and EMPOP were included in the evaluation. Haplogroups were determined for 92 diverse mtDNA haplotypes by the three tools based on four regions: the entire mitochondrial genome (mitogenome), control region (CR), hypervariable segment 1 and 2 (HVS1-2) regions, and HVS1 only. There were only two differences (out of 92) between all three tools when using the mitogenome, and in these instances the haplogroups were less precise by one or two nodes. Haplogroup assignments for the CR and HVS1-2 were similar; though less precise haplogroups resulted for these HVS1-2 haplotypes compared to the CR due to haplogroup-diagnostic mutations outside the queried region. As an important QC aspect, the comparison between the haplogroup predictions of authentic and artificial haplotypes showed that it is possible to identify recombinant mitogenome haplotypes from two large (~8500-bp) amplicons, but the differentiation is more difficult with HVS1-2 “artificial” haplotypes. Overall, the tools performed similarly, but EMPOP’s SAM2 produced more precise haplogroup predictions than AQME and HaploGrep2 across all haplogroups and regions. An important consideration when using any haplogrouping tool is that the haplogroup identified is only a prediction, particularly when based on smaller regions. All three tools provided valuable phylogenetic information to enable QC of mtDNA data