Evidence for multi-copy Mega-NUMTs in the human genome.

The maternal mode of mitochondrial DNA (mtDNA) inheritance is central to human genetics. Recently, evidence for bi-parental inheritance of mtDNA was claimed for individuals of three pedigrees that suffered mitochondrial disorders. We sequenced mtDNA using both direct Sanger and Massively Parallel Sequencing in several tissues of eleven maternally related and other affiliated healthy individuals of a family pedigree and observed mixed mitotypes in eight individuals. Cells without nuclear DNA, i.e. thrombocytes and hair shafts, only showed the mitotype of haplogroup (hg) V. Skin biopsies were prepared to generate ρ° cells void of mtDNA, sequencing of which resulted in a hg U4c1 mitotype. The position of the Mega-NUMT sequence was determined by fluorescence in situ hybridization and two different quantitative PCR assays were used to determine the number of contributing mtDNA copies. Thus, evidence for the presence of repetitive, full mitogenome Mega-NUMTs matching haplogroup U4c1 in various tissues of eight maternally related individuals was provided. Multi-copy Mega-NUMTs mimic mixtures of mtDNA that cannot be experimentally avoided and thus may appear in diverse fields of mtDNA research and diagnostics. We demonstrate that hair shaft mtDNA sequencing provides a simple but reliable approach to exclude NUMTs as source of misleading results.

Helena's Many Daughters: More Mitogenome Diversity behind the Most Common West Eurasian mtDNA Control Region Haplotype in an Extended Italian Population Sample.

The high number of matching haplotypes of the most common mitochondrial (mt)DNA lineages are considered to be the greatest limitation for forensic applications. This study investigates the potential to solve this constraint by massively parallel sequencing a large number of mitogenomes that share the most common West Eurasian mtDNA control region (CR) haplotype motif (263G 315.1C 16519C). We augmented a pilot study on 29 to a total of 216 Italian mitogenomes that represents the largest set of the most common CR haplotype compiled from a single country. The extended population sample confirmed and extended the huge coding region diversity behind the most common CR motif. Complete mitogenome sequencing allowed for the detection of 163 distinct haplotypes, raising the power of discrimination from 0 (CR) to 99.6% (mitogenome). The mtDNAs were clustered into 61 named clades of haplogroup H and did not reveal phylogeographic trends within Italy. Rapid individualization approaches for investigative purposes are limited to the most frequent H clades of the dataset, viz. H1, H3, and H7.

Human settlement history between Sunda and Sahul: a focus on East Timor (Timor-Leste) and the Pleistocenic mtDNA diversity.

BACKGROUND: Distinct, partly competing, "waves" have been proposed to explain human migration in(to) today's Island Southeast Asia and Australia based on genetic (and other) evidence. The paucity of high quality and high resolution data has impeded insights so far. In this study, one of the first in a forensic environment, we used the Ion Torrent Personal Genome Machine (PGM) for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice. RESULTS: In this first representative investigation on the mitochondrial DNA (mtDNA) variation of East Timor (Timor-Leste) population including >300 individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup P1, an indigenous lineage of the Southwest Pacific region. Our results suggest a colonization of southern Sahul (Australia) >37 kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern Sahul (New Guinea) followed by westward migrations <28 kya. CONCLUSIONS: The temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated "later" events of (South) East Asian origin pinpoints a highly dynamic migratory phase.

Rapid coastal spread of First Americans: novel insights from South America's Southern Cone mitochondrial genomes.

It is now widely agreed that the Native American founders originated from a Beringian source population ~15-18 thousand years ago (kya) and rapidly populated all of the New World, probably mainly following the Pacific coastal route. However, details about the migration into the Americas and the routes pursued on the continent still remain unresolved, despite numerous genetic, archaeological, and linguistic investigations. To examine the pioneering peopling phase of the South American continent, we screened literature and mtDNA databases and identified two novel mitochondrial DNA (mtDNA) clades, here named D1g and D1j, within the pan-American haplogroup D1. They both show overall rare occurrences but local high frequencies, and are essentially restricted to populations from the Southern Cone of South America (Chile and Argentina). We selected and completely sequenced 43 D1g and D1j mtDNA genomes applying highest quality standards. Molecular and phylogeographic analyses revealed extensive variation within each of the two clades and possibly distinct dispersal patterns. Their age estimates agree with the dating of the earliest archaeological sites in South America and indicate that the Paleo-Indian spread along the entire longitude of the American double continent might have taken even <2000 yr. This study confirms that major sampling and sequencing efforts are mandatory for uncovering all of the most basal variation in the Native American mtDNA haplogroups and for clarification of Paleo-Indian migrations, by targeting, if possible, both the general mixed population of national states and autochthonous Native American groups, especially in South America.

The initial peopling of the Americas: a growing number of founding mitochondrial genomes from Beringia.

Pan-American mitochondrial DNA (mtDNA) haplogroup C1 has been recently subdivided into three branches, two of which (C1b and C1c) are characterized by ages and geographical distributions that are indicative of an early arrival from Beringia with Paleo-Indians. In contrast, the estimated ages of C1d--the third subset of C1--looked too young to fit the above scenario. To define the origin of this enigmatic C1 branch, we completely sequenced 63 C1d mitochondrial genomes from a wide range of geographically diverse, mixed, and indigenous American populations. The revised phylogeny not only brings the age of C1d within the range of that of its two sister clades, but reveals that there were two C1d founder genomes for Paleo-Indians. Thus, the recognized maternal founding lineages of Native Americans are at least 15, indicating that the overall number of Beringian or Asian founder mitochondrial genomes will probably increase extensively when all Native American haplogroups reach the same level of phylogenetic and genomic resolution as obtained here for C1d.

Mitochondrial haplogroup U5b3: a distant echo of the epipaleolithic in Italy and the legacy of the early Sardinians.

There are extensive data indicating that some glacial refuge zones of southern Europe (Franco-Cantabria, Balkans, and Ukraine) were major genetic sources for the human recolonization of the continent at the beginning of the Holocene. Intriguingly, there is no genetic evidence that the refuge area located in the Italian Peninsula contributed to this process. Here we show, through phylogeographic analyses of mitochondrial DNA (mtDNA) variation performed at the highest level of molecular resolution (52 entire mitochondrial genomes), that the most likely homeland for U5b3-a haplogroup present at a very low frequency across Europe-was the Italian Peninsula. In contrast to mtDNA haplogroups that expanded from other refugia, the Holocene expansion of haplogroup U5b3 toward the North was restricted by the Alps and occurred only along the Mediterranean coasts, mainly toward nearby Provence (southern France). From there, approximately 7,000-9,000 years ago, a subclade of this haplogroup moved to Sardinia, possibly as a result of the obsidian trade that linked the two regions, leaving a distinctive signature in the modern people of the island. This scenario strikingly matches the age, distribution, and postulated geographic source of a Sardinian Y chromosome haplogroup (I2a2-M26), a paradigmatic case in the European context of a founder event marking both female and male lineages.

Accumulation of mutations over the entire mitochondrial genome of breast cancer cells obtained by tissue microdissection.

The occurrence of heteroplasmy and mixtures is technically challenging for the analysis of mitochondrial DNA. More than that, observed mutations need to be carefully interpreted in the light of the phylogeny as mitochondrial DNA is a uniparental marker reflecting human evolution. Earlier attempts to explain the role of mtDNA in cancerous tissues led to substantial confusion in medical genetics mainly due to the presentation of low sequence data quality and misinterpretation of mutations representing a particular haplogroup background rather than being cancer-specific. The focus of this study is to characterize the extent and level of mutations in breast cancer samples obtained by tissue microdissection by application of an evaluated full mtDNA genome sequencing protocol. We amplified and sequenced the complete mitochondrial genomes of microdissected breast cancer cells of 15 patients and compared the results to those obtained from paired non-cancerous breast tissue derived from the same patients. We observed differences in the heteroplasmic states of substitutions between cancerous and normal cells, one of which was affecting a position that has been previously reported in lung cancer and another one that has been identified in 16 epithelial ovarian tumors, possibly indicating functional relevance. In the coding region, we found full transitions in two cancerous mitochondrial genomes and 12 heteroplasmic substitutions as compared to the non-cancerous breast cells. We identified somatic mutations over the entire mtDNA of human breast cancer cells potentially impairing the mitochondrial OXPHOS system.

The Ancestry of Eastern Paraguay: A Typical South American Profile with a Unique Pattern of Admixture.

Immigrants from diverse origins have arrived in Paraguay and produced important demographic changes in a territory initially inhabited by indigenous Guarani. Few studies have been performed to estimate the proportion of Native ancestry that is still preserved in Paraguay and the role of females and males in admixture processes. Therefore, 548 individuals from eastern Paraguay were genotyped for three marker sets: mtDNA, Y-SNPs and autosomal AIM-InDels. A genetic homogeneity was found between departments for each set of markers, supported by the demographic data collected, which showed that only 43% of the individuals have the same birthplace as their parents. The results show a sex-biased intermarriage, with higher maternal than paternal Native American ancestry. Within the native mtDNA lineages in Paraguay (87.2% of the total), most haplogroups have a broad distribution across the subcontinent, and only few are concentrated around the Paraná River basin. The frequency distribution of the European paternal lineages in Paraguay (92.2% of the total) showed a major contribution from the Iberian region. In addition to the remaining legacy of the colonial period, the joint analysis of the different types of markers included in this study revealed the impact of post-war migrations on the current genetic background of Paraguay.

Increasing the discrimination power of forensic STR testing by employing high-performance mass spectrometry, as illustrated in indigenous South African and Central Asian populations.

Short tandem repeat (STR) typing has become the standard technique in forensic methodology for the identification of unknown samples. National DNA databases have been established that contain STR genotypes for intelligence purposes. Due to their success, national DNA databases have been growing so fast that the number of advantageous matches may become a logistic problem for the analysts. This is especially true for partial STR profiles as they display reduced discrimination power. To overcome this drawback, modified versions (so-called mini-STRs) of existing loci were introduced as well as new loci to improve the information content of (partial) STR profiles. We pursue an alternative approach that makes use of nucleotide variation within the amplified STR fragments, which can be discerned by mass spectrometry. We have developed an assay that determines molecular masses from crude STR amplicons which were purified and separated by a liquid chromatographic system directly hyphenated to an electrospray ionization mass spectrometer. We present here new population data of forensically relevant STRs in Khoisan and Yakut populations. These autochthonous groups were selected as they may harbor additional STR alleles that are rare or unobserved in modern humans from cosmopolitan areas, especially for the Khoisan, which are known to represent a very ancient human population. The analysis of the molecular mass of STRs offered a widened spectrum of allele variability escorted by enhanced forensic use. Thus, established STR data derived from fragment size analysis can still be used in casework or in the context of intelligence databasing.

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.

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.

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.

Evaluation of mitogenome sequence concordance, heteroplasmy detection, and haplogrouping in a worldwide lineage study using the Precision ID mtDNA Whole Genome Panel.

The emergence of Massively Parallel Sequencing technologies enabled the analysis of full mitochondrial (mt)DNA sequences from forensically relevant samples that have, so far, only been typed in the control region or its hypervariable segments. In this study, we evaluated the performance of a commercially available multiplex-PCR-based assay, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific), for the amplification and sequencing of the entire mitochondrial genome (mitogenome) from even degraded forensic specimens. For this purpose, more than 500 samples from 24 different populations were selected to cover the vast majority of established superhaplogroups. These are known to harbor different signature sequence motifs corresponding to their phylogenetic background that could have an effect on primer binding and, thus, could limit a broad application of this molecular genetic tool. The selected samples derived from various forensically relevant tissue sources and were DNA extracted using different methods. We evaluated sequence concordance and heteroplasmy detection and compared the findings to conventional Sanger sequencing as well as an orthogonal MPS platform. We discuss advantages and limitations of this approach with respect to forensic genetic workflow and analytical requirements.

Increased forensic efficiency of DNA fingerprints through simultaneous resolution of length and nucleotide variability by high-performance mass spectrometry.

Short tandem repeat (STR) typing is the most powerful method for determining the origin of a sample for a number of molecular disciplines such as medical genetics, population genetics, tumor analysis, transplantation medicine, or forensic crime scene analysis. STR alleles are routinely differentiated based upon their fragment size by electrophoresis under denaturing conditions, which does not take nucleotide variability into consideration. This simplification leads to loss of biological information as the nature of the individual sequence motifs that build an STR is not described. An alternative detection platform would be mass spectrometry, which captures the underlying sequence variation by comparing the molecular masses of DNA fragments. Here, we demonstrate that the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (ICEMS) is able to simultaneously detect length and nucleotide variability in STRs. Overall, 21 forensically relevant STRs that are also used in other scientific fields were screened in an Austrian population sample for the occurrence of nucleotide variability within or close to the repeat region. A total of 11 of the investigated loci (SE33, D2S1338, vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, and D2S441) brought additional allele (sequence) variants. Forensic efficiency, as determined by typical statistical parameters, was significantly increased by 20 to 30%. The beauty of ICEMS-STR-analysis is the fact that it represents one of the few technological advancements that allows direct comparison of newly generated data with existing data such as stored in DNA databases, which have a retarding effect on new developments.

Towards broadening Forensic DNA Phenotyping beyond pigmentation: Improving the prediction of head hair shape from DNA.

Human head hair shape, commonly classified as straight, wavy, curly or frizzy, is an attractive target for Forensic DNA Phenotyping and other applications of human appearance prediction from DNA such as in paleogenetics. The genetic knowledge underlying head hair shape variation was recently improved by the outcome of a series of genome-wide association and replication studies in a total of 26,964 subjects, highlighting 12 loci of which 8 were novel and introducing a prediction model for Europeans based on 14 SNPs. In the present study, we evaluated the capacity of DNA-based head hair shape prediction by investigating an extended set of candidate SNP predictors and by using an independent set of samples for model validation. Prediction model building was carried out in 9674 subjects (6068 from Europe, 2899 from Asia and 707 of admixed European and Asian ancestries), used previously, by considering a novel list of 90 candidate SNPs. For model validation, genotype and phenotype data were newly collected in 2415 independent subjects (2138 Europeans and 277 non-Europeans) by applying two targeted massively parallel sequencing platforms, Ion Torrent PGM and MiSeq, or the MassARRAY platform. A binomial model was developed to predict straight vs. non-straight hair based on 32 SNPs from 26 genetic loci we identified as significantly contributing to the model. This model achieved prediction accuracies, expressed as AUC, of 0.664 in Europeans and 0.789 in non-Europeans; the statistically significant difference was explained mostly by the effect of one EDAR SNP in non-Europeans. Considering sex and age, in addition to the SNPs, slightly and insignificantly increased the prediction accuracies (AUC of 0.680 and 0.800, respectively). Based on the sample size and candidate DNA markers investigated, this study provides the most robust, validated, and accurate statistical prediction models and SNP predictor marker sets currently available for predicting head hair shape from DNA, providing the next step towards broadening Forensic DNA Phenotyping beyond pigmentation traits.

Lack of gene-language correlation due to reciprocal female but directional male admixture in Austronesians and non-Austronesians of East Timor.

Nusa Tenggara, including East Timor, located at the crossroad between Island Southeast Asia, Near Oceania, and Australia, are characterized by a complex cultural structure harbouring speakers from two different major linguistic groups of different geographic origins (Austronesian (AN) and non-Austronesian (NAN)). This provides suitable possibilities to study gene-language relationship; however, previous studies from other parts of Nusa Tenggara reported conflicting evidence about gene-language correlation in this region. Aiming to investigate gene-language relationships including sex-mediated aspects in East Timor, we analysed the paternally inherited non-recombining part of the Y chromosome (NRY) and the maternally inherited mitochondrial (mt) DNA in a representative collection of AN- and NAN-speaking groups. Y-SNP (single-nucleotide polymorphism) data were newly generated for 273 samples and combined with previously established Y-STR (short tandem repeat) data of the same samples, and with previously established mtDNA data of 290 different samples with, however, very similar representation of geographic and linguistic coverage of the country. We found NRY and mtDNA haplogroups of previously described putative East/Southeast Asian (E/SEA) and Near Oceanian (NO) origins in both AN and NAN speakers of East Timor, albeit in different proportions, suggesting reciprocal genetic admixture between both linguistic groups for females, but directional admixture for males. Our data underline the dual genetic origin of East Timorese in E/SEA and NO, and highlight that substantial genetic admixture between the two major linguistic groups had occurred, more so via women than men. Our study therefore provides another example where languages and genes do not conform due to sex-biased genetic admixture across major linguistic groups.

Hairy matters: MtDNA quantity and sequence variation along and among human head hairs.

Hairs from the same donor have been found to differ in mtDNA sequence within and among themselves and from other tissues, which impacts interpretation of results obtained in a forensic setting. However, little is known on the magnitude of this phenomenon and published data on systematic studies are scarce. We addressed this issue by generating mtDNA control region (CR) profiles of >450 hair fragments from 21 donors by Sanger-type sequencing (STS). To mirror forensic scenarios, we compared hair haplotypes from the same donors to each other, to the corresponding buccal swab reference haplotypes and analyzed several fragments of individual hairs. We also investigated the effects of hair color, donor sex and age, mtDNA haplogroup and chemical treatment on mtDNA quantity, amplification success and variation. We observed a wide range of individual CR sequence variation. The reference haplotype was the only or most common (≥75%) hair haplotype for most donors. However, in two individuals, the reference haplotype was only found in about a third of the investigated hairs, mainly due to differences at highly variable positions. Similarly, most hairs revealed the reference haplotype along their entire length, however, about a fifth of the hairs contained up to 71% of segments with deviant haplotypes, independent of the longitudinal position. Variation affected numerous positions, typically restricted to the individual hair and in most cases heteroplasmic, but also fixed (i.e. homoplasmic) substitutions were observed. While existing forensic mtDNA interpretation guidelines were found still sufficient for all comparisons to reference haplotypes, some comparisons between hairs from the same donor could yield false exclusions when those guidelines are strictly followed. This study pinpoints the special care required when interpreting mtDNA results from hair in forensic casework.

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.

Mapping human dispersals into the Horn of Africa from Arabian Ice Age refugia using mitogenomes.

Rare mitochondrial lineages with relict distributions can sometimes be disproportionately informative about deep events in human prehistory. We have studied one such lineage, haplogroup R0a, which uniquely is most frequent in Arabia and the Horn of Africa, but is distributed much more widely, from Europe to India. We conclude that: (1) the lineage ancestral to R0a is more ancient than previously thought, with a relict distribution across the Mediterranean/Southwest Asia; (2) R0a has a much deeper presence in Arabia than previously thought, highlighting the role of at least one Pleistocene glacial refugium, perhaps on the Red Sea plains; (3) the main episode of dispersal into Eastern Africa, at least concerning maternal lineages, was at the end of the Late Glacial, due to major expansions from one or more refugia in Arabia; (4) there was likely a minor Late Glacial/early postglacial dispersal from Arabia through the Levant and into Europe, possibly alongside other lineages from a Levantine refugium; and (5) the presence of R0a in Southwest Arabia in the Holocene at the nexus of a trading network that developed after ~3 ka between Africa and the Indian Ocean led to some gene flow even further afield, into Iran, Pakistan and India.

Helena, the hidden beauty: Resolving the most common West Eurasian mtDNA control region haplotype by massively parallel sequencing an Italian population sample.

The analysis of mitochondrial (mt)DNA is a powerful tool in forensic genetics when nuclear markers fail to give results or maternal relatedness is investigated. The mtDNA control region (CR) contains highly condensed variation and is therefore routinely typed. Some samples exhibit an identical haplotype in this restricted range. Thus, they convey only weak evidence in forensic queries and limited phylogenetic information. However, a CR match does not imply that also the mtDNA coding regions are identical or samples belong to the same phylogenetic lineage. This is especially the case for the most frequent West Eurasian CR haplotype 263G 315.1C 16519C, which is observed in various clades within haplogroup H and occurs at a frequency of 3-4% in many European populations. In this study, we investigated the power of massively parallel complete mtGenome sequencing in 29 Italian samples displaying the most common West Eurasian CR haplotype - and found an unexpected high diversity. Twenty-eight different haplotypes falling into 19 described sub-clades of haplogroup H were revealed in the samples with identical CR sequences. This study demonstrates the benefit of complete mtGenome sequencing for forensic applications to enforce maximum discrimination, more comprehensive heteroplasmy detection, as well as highest phylogenetic resolution.