Development and expansion of high-quality control region databases to improve forensic mtDNA evidence interpretation

In an effort to increase the quantity, breadth and availability of mtDNA databases suitable for forensic comparisons, we have developed a high-throughput process to generate approximately 5000 control region sequences per year from regional US populations, global populations from which the current US population is derived and global populations currently under-represented in available forensic databases. The system utilizes robotic instrumentation for all laboratory steps from pre-extraction through sequence detection, and a rigorous eight-step, multi-laboratory data review process with entirely electronic data transfer. Over the past 3 years, nearly 10,000 control region sequences have been generated using this approach. These data are being made publicly available and should further address the need for consistent, high-quality mtDNA databases for forensic testing.     

Hungarian mtDNA population databases from Budapest and the Baranya county Roma

To facilitate forensic mtDNA testing in Hungary, we have generated control region databases for two Hungarian populations: 211 individuals were sampled from the urban Budapest population and 208 individuals were sampled from a Romani (“gypsy”) population in Baranya county. Sequences were generated using a highly redundant approach to minimize potential database errors. The Budapest population had high sequence diversity with 180 lineages, 183 polymorphic positions, and a random match probability of 1%. In contrast, the Romani population exhibited low sequence diversity, with only 56 lineages, 109 segregating sites, and a random match probability of 8.8%. The mtDNA haplogroup compositions of the two populations were also distinct, with the large proportion of haplogroup M samples (35%) in the Roma the most obvious difference between the two populations. These factors highlight the importance of considering population structure when generating reference databases for forensic testing purposes. Comparisons between our Romani population sample and other published data indicate the need for heightened caution when sampling and using mtDNA databases of small endogamous populations. The Romani populations that we compared showed significant departures from genetic uniformity. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

HVI and HVII mitochondrial DNA data in Apaches and Navajos

Most mtDNA studies on Native Americans have concentrated on hypervariable region I (HVI) sequence data. Mitochondrial DNA haplotype data from hypervariable regions I and II (HVI and HVII) have been compiled from Apaches (N=180) and Navajos (N=146). The inclusion of HVII data increases the amount of information that can be obtained from low diversity population groups. Less mtDNA variation was observed in the Apaches and Navajos than in major population groups. The majority of the mtDNA sequences were observed more than once; only 17.8% (32/180) of the Apache sequences and 25.8% of the Navajo sequences were observed once. Most of the haplotypes in Apaches and Navajos fall into the A and B haplogroups. Although a limited number of haplogroups were observed, both sample populations exhibit sufficient variation for forensic mtDNA typing. Genetic diversity was 0.930 in the Apache sample and 0.963 in the Navajo sample. The random match probability was 7.48% in the Apache sample and 4.40% in the Navajo sample. The average number of nucleotide differences between individuals in a database is 9.0 in the Navajo sample and 7.7 in the Apache sample. The data demonstrate that mtDNA sequencing can be informative in forensic cases where Native American population data are used.     

Haplogrouping mitochondrial DNA sequences in Legal Medicine/Forensic Genetics

Haplogrouping refers to the classification of (partial) mitochondrial DNA (mtDNA) sequences into haplogroups using the current knowledge of the worldwide mtDNA phylogeny. Haplogroup assignment of mtDNA control-region sequences assists in the focused comparison with closely related complete mtDNA sequences and thus serves two main goals in forensic genetics: first is the a posteriori quality analysis of sequencing results and second is the prediction of relevant coding-region sites for confirmation or further refinement of haplogroup status. The latter may be important in forensic casework where discrimination power needs to be as high as possible. However, most articles published in forensic genetics perform haplogrouping only in a rudimentary or incorrect way. The present study features PhyloTree as the key tool for assigning control-region sequences to haplogroups and elaborates on additional Web-based searches for finding near-matches with complete mtDNA genomes in the databases. In contrast, none of the automated haplogrouping tools available can yet compete with manual haplogrouping using PhyloTree plus additional Web-based searches, especially when confronted with artificial recombinants still present in forensic mtDNA datasets. We review and classify the various attempts at haplogrouping by using a multiplex approach or relying on automated haplogrouping. Furthermore, we re-examine a few articles in forensic journals providing mtDNA population data where appropriate haplogrouping following PhyloTree immediately highlights several kinds of sequence errors.     

Establishing a database of Canadian feline mitotypes for forensic use

Hair shed by pet animals is often found and collected as evidence from crime scenes. Due to limitations such as small amount and low quality, mitochondrial DNA (mtDNA) is often the only type of DNA that can be used for linking the hair to a potential contributor. mtDNA has lower discriminatory power than nuclear DNA because multiple, unrelated individuals within a population can have the same mtDNA sequence, or mitotype. Therefore, to determine the evidentiary value of a match between crime scene evidence and a suspected contributor, the frequency of the mitotype must be known within the regional population. While mitotype frequencies have been determined for the United States’ cat population, the frequencies are unknown for the Canadian cat population. Given the countries’ close proximity and similar human settlement patterns, these populations may be homogenous, meaning a single, regional database may be used for estimating cat population mitotype frequencies. Here we determined the mitotype frequencies of the Canadian cat population and compared them to the United States’ cat population. The two cat populations are statistically homogenous, however mitotype B6 was found in high frequency in Canada and extremely low frequency in the United States, meaning a single database would not be appropriate for North America. Furthermore, this work calls attention to these local spikes in frequency of otherwise rare mitotypes, instances of which exist around the world and have the potential to misrepresent the evidentiary value of matches compared to a regional database.     

DNA Commission of the International Society for Forensic Genetics: Revised and extended guidelines for mitochondrial DNA typing

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the question of human identification. Previous recommendations published in 2000 addressed the analysis and interpretation of mitochondrial DNA (mtDNA) in forensic casework. While the foundations set forth in the earlier recommendations still apply, new approaches to the quality control, alignment and nomenclature of mitochondrial sequences, as well as the establishment of mtDNA reference population databases, have been developed. Here, we describe these developments and discuss their application to both mtDNA casework and mtDNA reference population databasing applications. While the generation of mtDNA for forensic casework has always been guided by specific standards, it is now well-established that data of the same quality are required for the mtDNA reference population data used to assess the statistical weight of the evidence. As a result, we introduce guidelines regarding sequence generation, as well as quality control measures based on the known worldwide mtDNA phylogeny, that can be applied to ensure the highest quality population data possible. For both casework and reference population databasing applications, the alignment and nomenclature of haplotypes is revised here and the phylogenetic alignment proffered as acceptable standard. In addition, the interpretation of heteroplasmy in the forensic context is updated, and the utility of alignment-free database searches for unbiased probability estimates is highlighted. Finally, we discuss statistical issues and define minimal standards for mtDNA database searches.     

mtDNA sequence diversity of Hazara ethnic group from Pakistan

The present study was undertaken to investigate mitochondrial DNA (mtDNA) control region sequences of Hazaras from Pakistan, so as to generate mtDNA reference database for forensic casework in Pakistan and to analyze phylogenetic relationship of this particular ethnic group with geographically proximal populations. Complete mtDNA control region (nt 16024-576) sequences were generated through Sanger Sequencing for 319 Hazara individuals from Quetta, Baluchistan. The population sample set showed a total of 189 distinct haplotypes, belonging mainly to West Eurasian (51.72%), East & Southeast Asian (29.78%) and South Asian (18.50%) haplogroups. Compared with other populations from Pakistan, the Hazara population had a relatively high haplotype diversity (0.9945) and a lower random match probability (0.0085). The dataset has been incorporated into EMPOP database under accession number EMP00680. The data herein comprises the largest, and likely most thoroughly examined, control region mtDNA dataset from Hazaras of Pakistan.     

MtDNA diversity of Ghana: a forensic and phylogeographic view

West Africa is characterized by a migration history spanning more than 150,000 years. Climate changes but also political circumstances were responsible for several early but also recent population movements that shaped the West African mitochondrial landscape. The aim of the study was to establish a Ghanaian mtDNA dataset for forensic purposes and to investigate the diversity of the Ghanaian population sample with respect to surrounding populations. We sequenced full mitochondrial control regions of 193 Akan people from Ghana and excluded two apparently close maternally related individuals due to preceding kinship testing. The remaining dataset comprising 191 sequences was applied as etalon for quasi-median network analysis and was subsequently combined with 99 additional control region sequences from surrounding West African countries. All sequences were incorporated into the EMPOP database enriching the severely underrepresented African mtDNA pool. For phylogeographic considerations, the Ghanaian haplotypes were compared to those of 19 neighboring populations comprising a total number of 6198 HVS1 haplotypes. We found extensive genetic admixture between the Ghanaian lineages and those from adjacent populations diminishing with geographical distance. The extent of genetic admixture reflects the long but also recent history of migration waves within West Africa mainly caused by changing environmental conditions. Also, evidence for potential socio-economical influences such as trade routes is provided by the occurrence of U6b and U6d sequences found in Dubai but also in Tunisia leading to the African West Coast via Mauritania and Senegal but also via Niger, Nigeria to Cameroon.     

Forensic and phylogeographic characterization of mtDNA lineages from northern Thailand (Chiang Mai)

The immigration of diverse ethnic groups over the past centuries from surrounding countries into Thailand left footprints in the genetic composition of Thai mitochondrial DNA (mtDNA) lineages. The entire mtDNA control region (1,122 bp) was typed in 190 unrelated male volunteers from the northern Thailand province of Chiang Mai following highest quality standards. For a more precise haplogroup classification, selected single nucleotide polymorphisms from the mtDNA coding region were genotyped. We found several new, so far undescribed mtDNA lineages. Quasi-median networks were constructed for visualisation of character conflicts. The data were put into population-genetic relationships with other Southeast Asian populations. Although the frequencies of the Thai haplogroups were characteristic for Southeast Asia in terms of haplotype composition and genetic structure, the Thai population was significantly different from other Southeast Asian populations. This necessitates establishing regional databases, especially for forensic applications. The population data have been submitted to the EMPOP database () and will be available on publication.     

Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population

The forensic application of mitochondrial DNA (mtDNA) typing requires large and regionally well-defined databases. To expand the database for forensic identification purposes in Slovenia, the mtDNA control region sequences of the hypervariable regions HVI and HVII were determined in a population of 129 maternally unrelated Slovenians, using a fluorescent-based capillary electrophoresis sequencing method. A total of 111 different haplotypes resulting from 124 polymorphic positions (80 polymorphic positions in HVI and 44 in HVII) were found. Of these, 101 mtDNA types were unique, 6 haplotypes were shared by 2 individuals, 1 haplotype by 3 individuals, 2 haplotypes by 4 individuals, and the most common haplotype was found in 5 individuals. The most frequent haplotypes in the Slovenian population ,263(G), 315.1(C) and 263(G), 309.1(C), 315.1(C) are also the most common in other European populations. The data support the concept that these haplotypes may represent a common European mtDNA sequence types. The sequence poymorphisms were compared to the databases of west Austria and central Italy and the HVI and HVII sequence matching probabilities within and between populations were calculated. It is 1.1–4.5 times more likely to find a sequence match in a random pair of Slovenians than in a random Slovenian-Italian pair and in a random Slovenian-Austrian pair. The length heteroplasmy in the homopolymeric C-stretch regions located at nucleotide positions 16184–16193 in HVI and at positions 303–315 in HVII was observed in 17% and 8% of individuals, respectively. A statistical estimate of the results for this population showed the random match probability and the genetic diversity of 1.16% and 0.996, respectively.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00414-003-0394-3     

Social benefits of non-criminal genetic databases: missing persons and human remains identification

A Missing Persons Genetic Identification Program (Phoenix Program) was implemented in Spain in order to try to identify cadavers and human remains that could not be identified using traditional forensic approaches; to our knowledge, this is the first database ever implemented and in function in the world. Two separate mitochondrial DNA (mtDNA) databases have been generated and comparisons can be made automatically to match identical or similar sequences contained in both databases. One database is called the Reference Database (RD), which contains mtDNA sequences from maternal relatives of missing persons that provide the samples voluntarily after informed consent. The other database is called the Questioned Database (QD) and is comprised of mtDNA data on unknown remains and cadavers that could not be unequivocally identified. The combined database is a civil database designed solely for human identification and because of the informed consent and voluntary donation of reference samples is different from other databases now used to solve criminal cases. It is timely and incumbent on other willing countries to begin an international collaboration so compatibility and full utility can be enjoyed with this kind of non-criminal database. Received: 21 November 2000 / Accepted: 19 July 2001     

Amerindian mitochondrial DNA haplogroups predominate in the population of Argentina: towards a first nationwide forensic mitochondrial DNA sequence database

The study presents South American mitochondrial DNA (mtDNA) data from selected north (N = 98), central (N = 193) and south (N = 47) Argentinean populations. Sequence analysis of the complete mtDNA control region (CR, 16024–576) resulted in 288 unique haplotypes ignoring C-insertions around positions 16193, 309, and 573; the additional analysis of coding region single nucleotide polymorphisms enabled a fine classification of the described lineages. The Amerindian haplogroups were most frequent in the north and south representing more than 60% of the sequences. A slightly different situation was observed in central Argentina where the Amerindian haplogroups represented less than 50%, and the European contribution was more relevant. Particular clades of the Amerindian subhaplogroups turned out to be nearly region-specific. A minor contribution of African lineages was observed throughout the country. This comprehensive admixture of worldwide mtDNA lineages and the regional specificity of certain clades in the Argentinean population underscore the necessity of carefully selecting regional samples in order to develop a nationwide mtDNA database for forensic and anthropological purposes. The mtDNA sequencing and analysis were performed under EMPOP guidelines in order to attain high quality for the mtDNA database.     

Bedeutung der mtDNA-Analyse für forensische Fragestellungen

The analysis of mitochondrial DNA (mtDNA) represents a technological niche in forensic cases where samples have to be identified that contain only nuclear DNA of limited quality and quantity. The high copy number, the increased stability against degradation and the strict maternal inheritance are the main characteristics of the mitochondrial genome which makes it particularly suitable for palaeogenetic inferences and the reconstruction of human evolution. In forensic analyses mtDNA profiling is an established technological resource for cases where conventional nuclear DNA markers fail to give satisfactory results (e.g. analyses of hair shafts, remains of bones and teeth). Substantial collections of validated mtDNA sequences are essential for a meaningful biostatistical evaluation of mtDNA profiles in a given case as the relative frequencies of the haplotypes can only be determined from adequate mtDNA databases. As mtDNA is inherited along a phylogeny, evolutionary features are also important for the interpretation of the data in individual case examples.     

The genetic legacy of the Transatlantic Slave Trade in the island of New Providence

The Bahamian archipelago has been influenced by a wide array of settlers (Lucayans, Eleutherian Adventurers, British Loyalists, Creoles from the United States and African slaves) throughout its short but dynamic history. Nevertheless, the Bahamas remains poorly characterized genetically and little is known about each group's contribution to the island chain. In the current study, the population of New Providence was analyzed based on 15 autosomal STR loci routinely employed in forensic DNA fingerprinting applications. A comparison of this collection with African groups reveals similar genetic profiles to West African populations from Equatorial Guinea and Angola, possibly resulting from the importation of slaves from West African ports during the Transatlantic Slave Trade. Although the New Providence collection exhibits strong genetic affinities to the two US African American reference populations, the detection of unique alleles among them may necessitate the utilization of population-specific databases in forensic cases especially when the STR profiles include these specific variants.     

Mitochondrial DNA control region variation in an autochthonous Basque population sample from the Basque Country

Mitochondrial control region (16024-576) sequences were generated from 106 samples from autochthonous Basques from the Autonomous Community of the Basque Country. It is especially important to generate mtDNA databases from isolated populations in order to maximize the power of discrimination of this molecular marker. It also represents a useful approach to carry out a more accurate haplogroup classification. This is the first database report of complete control region sequences in an autochthonous Basque population sample. Strict selection criteria of autochthonous individuals, automation of laboratory processing and independent reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data of the autochthonous Basque population.     

Fehlerquellen mitochondrialer DNS-Datensätze und Evaluation der mtDNS-Datenbank „D-Loop-BASE“

The analysis of mitochondrial (mt)DNA has become a technological niche in forensic casework, which is a challenging task for laboratories, both with regard to laboratory routine and data validation and interpretation. A posteriori analyses of mitochondrial data sets as well as recent systematic research during proficiency testing, have shown that especially the anonymized analysis of a large number of samples (as for example in the process of setting-up mtDNA population databases) is accompanied by a high risk of error. It has become apparent that not so much chemical factors or the technical equipment are to blame, but that these mistakes are usually to be traced to human error (clerical mistakes, mix-ups and false interpretation). A heightened sensibility with regard to this phenomenon is advisable particularly in forensic science, where high quality standards are essential. The project “D-Loop-BASE”, a central European database for non-coding mitochondrial sequences, was introduced in Munich at the ‘21st Workshop’ of the ‘Deutsche Gesellschaft für Rechtsmedizin’. One particular incident (test inquiry to D-Loop-BASE) raised doubts as to whether the database was functional and could produce meaningful results. The initial findings revealed errors in conception and programming. The D-Loop-BASE contained both inaccurate published data as well as unpublished and published incorrect sequences.     

A new view on the European feline population from mtDNA analysis in Polish domestic cats

Domestic cats from Eastern Europe have been poorly represented in studies on mitochondrial DNA diversity for forensic purposes until now. The aim of the present study was to contribute to closing this gap. The genetic structure and the origin of a cat population in Poland were examined against the background of human migrations over the centuries. One hundred and eighty-one cats from animal shelters in seven cities were genotyped. Twenty-one mtDNA haplotypes were found, with only one haplotype present in each of the populations, at an average frequency of 63.54%, and 13 haplotypes being found only in single populations. The analysis revealed the unexpectedly high frequency of haplotype PL02, in previous studies observed only in single cats. Differences in the number of the haplotypes, from four to eight, were observed among the shelters. The findings are discussed with regard to a world-wide database of feline sequences and to the complicated history of Poland. The study underscores the necessity of creating local databases of haplotypes that are of high evidentiary value to the forensic investigations conducted in a given country.     

Sequenzanalyse mitochondrialer DNA in der Rechtsmedizin

Over the last decade mtDNA analysis has gained high importance for forensic identification casework, archeological research as well as for evolutionary and population genetic studies. The structure of the mitochondrial DNA is described with particular consideration of the non-coding hypervariable regions. Furthermore the importance of ¶sequence polymorphisms for evolution biological investigations is discussed. The forensic value of the size and the stucture of sequence databases is underlined. Experimental studies dealing with mtDNA analysis of extracts from teeth, hair shafts and contaminated evidential material are presented. Finally three forensic criminal cases solved by mtDNA analysis are reported.