Post hoc deconvolution of human mitochondrial DNA mixtures by EMMA 2 using fine-tuned Phylotree nomenclature.

In this paper we present a new algorithm for splitting (partial) human mitogenomes into components with high similarity to haplogroup motifs of Phylotree. The algorithm reads a (partial) mitogenome coded by the differences to the reference (rCRS) and outputs the estimated haplogroups of the putative components. The algorithm requires no special information on the raw data of the sequencing process and is therefore suited for the post hoc analysis of mixtures of any sequencing technology. The software EMMA 2 implementing the algorithm will be made available via the EMPOP (https://empop.online) database and extends the nine years old software EMMA for haplogrouping single mitogenomes to mixtures with at most three components.

The Mitochondrial DNA Landscape of Modern Mexico.

Mexico is a rich source for anthropological and population genetic studies with high diversity in ethnic and linguistic groups. The country witnessed the rise and fall of major civilizations, including the Maya and Aztec, but resulting from European colonization, the population landscape has dramatically changed. Today, the majority of Mexicans do not identify themselves as Indigenous but as admixed, and appear to have very little in common with their pre-Columbian predecessors. However, when the maternally inherited mitochondrial (mt)DNA is investigated in the modern Mexican population, this is not the case. Control region sequences of 2021 samples deriving from all over the country revealed an overwhelming Indigenous American legacy, with almost 90% of mtDNAs belonging to the four major pan-American haplogroups A2, B2, C1, and D1. This finding supports a very low European contribution to the Mexican gene pool by female colonizers and confirms the effectiveness of employing uniparental markers as a tool to reconstruct a country's history. In addition, the distinct frequency and dispersal patterns of Indigenous American and West Eurasian clades highlight the benefit such large and country-wide databases provide for studying the impact of colonialism from a female perspective and population stratification. The importance of geographical database subsets not only for forensic application is clearly demonstrated.

Mitochondrial DNA control region variation in an Iraqi population sample.

BACKGROUND: Although Iraq has one of the world's oldest cultural histories and an important geographic location, forensic reference data on mitochondrial DNA (mtDNA) control region in Iraqi populations are scarce, particularly for populations residing in the southern part of Iraq. Mitochondrial DNA typing is an excellent tool for forensic investigations and in missing-person cases because of its unique qualities, such as mtDNA non-coding control region with specific genetic markers, high copy numbers in cells, maternal inheritance, and lack of recombination. METHODS: Forensic analysis was performed on the entire mtDNA control region in 203 unrelated Iraqi individuals residing in Samawah City of Iraq. Polymorphisms in the mtDNA were detected using polymerase chain reaction and Sanger-type sequencing, and the sequences were aligned to compare with revised Cambridge Reference Sequence (rCRS). RESULTS: The sequencing results revealed 111 haplotypes characterized by 143 polymorphic positions. Of these haplotypes, 63 were unique and 48 were shared by more than one person. The haplotype data generated in this study will be available on EMPOP via accession number EMP00814.

Pathogenic Variant Filtering for Mitochondrial Genome Haplotype Reporting.

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.

Mitochondrial DNA variation in Sub-Saharan Africa: Forensic data from a mixed West African sample, Côte d'Ivoire (Ivory Coast), and Rwanda.

This study provides 398 novel complete mitochondrial control region sequences that augment the still underrepresented data from Africa by three datasets: a mixed West African sample set deriving from 12 countries (n = 145) and datasets from Côte d'Ivoire (Ivory Coast) (n = 100) as well as Rwanda (n = 153). The analysis of mtDNA variation and genetic comparisons with published data revealed low random match probabilities in all three datasets and typical West African and East African diversity, respectively. Genetic parameters indicate that the presented mixed West African dataset may serve as first forensic mtDNA control region database for West Africa in general. In addition, a strategy for responsible forensic application of precious mtDNA population samples potentially containing close maternal relatives is outlined. The datasets will be uploaded to the forensic mtDNA database EMPOP (https://empop.online) upon publication.

Mitochondrial DNA control region variation in Lebanon, Jordan, and Bahrain.

This study investigated the mitochondrial DNA (mtDNA) control region variation in Middle Eastern populations (610 individuals from Lebanon, Jordan and the Kingdom of Bahrain) for which population data are scarce. FST comparison among populations revealed that there are significant differences in mtDNA distributions between Bahrain and the two other populations, while Lebanon and Jordan showed no significant differences. This was also reflected by the distribution of the observed lineages that differed prominently between Bahrain and the other two investigated populations. Jordan and Lebanon fit the hitherto known genetic results of the Levant population. Data are available via EMPOP (https://empop.online) and GenBank.

The maternal inheritance of Alto Paraná revealed by full mitogenome sequences.

Most studies on maternal lineages of South America populations are restricted to control region (CR) markers and, for some geographical regions, the number of studied samples does not adequately represent the existing diversity. This is the case of mitochondrial DNA (mtDNA) studies on Paraguay that are limited to two Native ethnic groups. To overcome this deficiency, we analysed the mitogenomes from 105 individuals living in Alto Paraná, the second most populated department of the country. Using the Precision ID mtDNA Whole Genome Panel, the molecule was sequenced on Ion S5. The majority of the haplotypes belong to the Native American lineages A, B, C and D. Analyses of maximum parsimony using mitogenome data retrieved from publications and in The 1000 Genomes Project showed a high number of new native American subclades in Paraguay. Also, none of the haplotypes found in Alto Paraná match the remaining South American samples, which include admixed populations from Colombia, Peru and Ecuador, and natives from Colombia and Ecuador. FST genetic distance analysis showed that the native genetic background of Alto Paraná has an intermediate position between the Amazonian groups and the admixed populations from Peru and Ecuador, supporting the theory about the Amazonian origin of the Tupi-Guarani and, at the same time, showing the influence of other linguistic groups.

Next generation database search algorithm for forensic mitogenome analyses.

Mitochondrial DNA (mtDNA) variation is being reported relative to the corrected version of the first sequenced human mitochondrial genome. A review of the existing literature across disciplines that employ mtDNA demonstrates that insertions and deletions are not reported in a standardized way. This may lead to false exclusions of identical sequences, unidentified matches in missing persons mtDNA databases, biased mtDNA database frequency estimates and overestimation of the genetic evidence. Seven years ago we introduced alignment-free database search software (SAM) and implemented it into the mtDNA database EMPOP (https://empop.online) to produce reliable and conservative frequency estimates that are required in the forensic context. However, ambiguity remained in how laboratories have been reporting mitotypes, as often more than one single alignment of a given mtDNA sequence was feasible. In order to overcome this limitation we here describe a concept and provide software for producing stable, harmonized phylogenetic alignment of mtDNA sequences for database searches. The new software SAM 2 will be made available via EMPOP and provide the user with the already established conservative frequency estimates. In addition, SAM 2 offers the rCRS-coded haplotype of a given mtDNA sequence following the established and widely accepted phylogenetic alignment. This provides the user with feedback on how mitotypes are stored in EMPOP and how they should be reported in order to harmonize nomenclature. Finally, this approach does not only permit reliable mtDNA nomenclature in forensics but invites related disciplines to take advantage of a standardized way of reporting mtDNA variation, thus closing the ranks between different genetic fields and supporting dialogue and collaboration between mtDNA scholars from various disciplines.

Updating the African human mitochondrial DNA tree: Relevance to forensic and population genetics.

Analysis of human mitochondrial DNA (mtDNA) variation plays an important role in forensic genetic investigations, especially in degraded biological samples and hair shafts. There are many issues of the mtDNA phylogeny that are of special interest to the forensic community, such as haplogroup classification or the post hoc investigation of potential errors in mtDNA datasets. We have analyzed >2200 mitogenomes of African ancestry with the aim of improving the known worldwide phylogeny. More than 300 new minor subclades were identified, and the Time to the Most Recent Common Ancestor (TMRCA) was estimated for each node of the phylogeny. Phylogeographic details are provided which might also be relevant to forensic genetics. The present study has special interest for forensic investigations because current analysis and interpretation of mtDNA casework rest on a solid worldwide phylogeny, as is evident from the role that phylogeny plays in popular resources in the field (e.g. PhyloTree), software (e.g. Haplogrep 2), and databases (e.g. EMPOP). Apart from this forensic genetic interest, we also highlight the impact of this research in anthropological studies, such as those related to the reconstruction of the transatlantic slave trade.

Mitochondrial DNA control region haplotype and haplogroup diversity in South Eastern Turkey.

Despite its large geographic and population size only little is known about the mitochondrial (mt)DNA make up of Turkey.orensically relevant data are almost completely absent in the literature. We analyzed the mtDNA control region of 224 volunteers from South Eastern Turkey and compared the data to populations from neighboring countries. The haplotypes will be made available via the EMPOP database (EMP00670) and contribute to the body of forensic mtDNA data.

High-quality mtDNA control region sequences from 680 individuals sampled across the Netherlands to establish a national forensic mtDNA reference database.

The use of mitochondrial DNA (mtDNA) for maternal lineage identification often marks the last resort when investigating forensic and missing-person cases involving highly degraded biological materials. As with all comparative DNA testing, a match between evidence and reference sample requires a statistical interpretation, for which high-quality mtDNA population frequency data are crucial. Here, we determined, under high quality standards, the complete mtDNA control-region sequences of 680 individuals from across the Netherlands sampled at 54 sites, covering the entire country with 10 geographic sub-regions. The complete mtDNA control region (nucleotide positions 16,024-16,569 and 1-576) was amplified with two PCR primers and sequenced with ten different sequencing primers using the EMPOP protocol. Haplotype diversity of the entire sample set was very high at 99.63% and, accordingly, the random-match probability was 0.37%. No population substructure within the Netherlands was detected with our dataset. Phylogenetic analyses were performed to determine mtDNA haplogroups. Inclusion of these high-quality data in the EMPOP database (accession number: EMP00666) will improve its overall data content and geographic coverage in the interest of all EMPOP users worldwide. Moreover, this dataset will serve as (the start of) a national reference database for mtDNA applications in forensic and missing person casework in the Netherlands.

Molecular genetic analysis on the remains of the Dark Countess: Revisiting the French Royal family.

The "Dark Counts" were a mysterious couple that appeared in the Thuringian village Eishausen in 1807. After living in self imposed solitude for 30 years the woman died and was buried under the name Sophia Botta. Her companion, who presented himself as Vavel de Versay, died in 1845 and was later identified as Leonardus Cornelius van der Valck, secretary of the Dutch embassy in Paris. Their lifestyle led to speculations that she was the true princess Marie Thérèse Charlotte of France, daughter of Louis XVI and Marie Antoinette. According to these speculations she was substituted by another young woman on a voyage from Paris to Vienna. Molecular genetic analyses were set out to test the remains attributed to the Dark Countess. Mitochondrial DNA testing brought concordant results determined in two forensic laboratories (Innsbruck, Austria and Freiburg, Germany) on parallel samples of the remains. The results were in exclusion to both, the mitochondrial lineage earlier reported for the French Royal family and the mitochondrial haplotype observed in a living descendant of the Royal family.

Mitochondrial DNA control region analysis of three ethnic groups in the Republic of Macedonia.

A total of 444 individuals representing three ethnic groups (Albanians, Turks and Romanies) in the Republic of Macedonia were sequenced in the mitochondrial control region. The mtDNA haplogroup composition differed between the three groups. Our results showed relatively high frequencies of haplogroup H12 in Albanians (8.8%) and less in Turks (3.3%), while haplogroups M5a1 and H7a1a were dominant in Romanies (13.7% and 10.3%, respectively) but rare in the former two. This highlights the importance of regional sampling for forensic mtDNA databasing purposes. These population data will be available on EMPOP under accession numbers EMP00644 (Albanians), EMP00645 (Romanies) and EMP00646 (Turks).

Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA).

The assignment of haplogroups to mitochondrial DNA haplotypes contributes substantial value for quality control, not only in forensic genetics but also in population and medical genetics. The availability of Phylotree, a widely accepted phylogenetic tree of human mitochondrial DNA lineages, led to the development of several (semi-)automated software solutions for haplogrouping. However, currently existing haplogrouping tools only make use of haplogroup-defining mutations, whereas private mutations (beyond the haplogroup level) can be additionally informative allowing for enhanced haplogroup assignment. This is especially relevant in the case of (partial) control region sequences, which are mainly used in forensics. The present study makes three major contributions toward a more reliable, semi-automated estimation of mitochondrial haplogroups. First, a quality-controlled database consisting of 14,990 full mtGenomes downloaded from GenBank was compiled. Together with Phylotree, these mtGenomes serve as a reference database for haplogroup estimates. Second, the concept of fluctuation rates, i.e. a maximum likelihood estimation of the stability of mutations based on 19,171 full control region haplotypes for which raw lane data is available, is presented. Finally, an algorithm for estimating the haplogroup of an mtDNA sequence based on the combined database of full mtGenomes and Phylotree, which also incorporates the empirically determined fluctuation rates, is brought forward. On the basis of examples from the literature and EMPOP, the algorithm is not only validated, but both the strength of this approach and its utility for quality control of mitochondrial haplotypes is also demonstrated.