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.

Distinguishing mitochondrial DNA and NUMT sequences amplified with the precision ID mtDNA whole genome panel.

Nuclear mitochondrial DNA segments (NUMTs) are generated via transfer of portions of the mitochondrial genome into the nuclear genome. Given their common origin, there is the possibility that both the mitochondrial and NUMT segments may co-amplify using the same set of primers. Thus, analysis of the variation of the mitochondrial genome must take into account this co-amplification of mitochondrial and NUMT sequences. The study herein builds on data from the study by Strobl et al. (Strobl et al., 2019), in which multiple point heteroplasmies were called with an "N" to prevent labeling NUMT sequences mimicking mitochondrial heteroplasmy and being interpreted as true mitochondrial in origin sequence variants. Each of these point heteroplasmies was studied in greater detail, both molecularly and bioinformatically, to determine whether NUMT or true mitochondrial DNA variation was present. The bioinformatic and molecular tools available to help distinguish between NUMT and mitochondrial DNA and the effect of NUMT sequences on interpretation were discussed.

The mitogenome portrait of Umbria in Central Italy as depicted by contemporary inhabitants and pre-Roman remains.

Umbria is located in Central Italy and took the name from its ancient inhabitants, the Umbri, whose origins are still debated. Here, we investigated the mitochondrial DNA (mtDNA) variation of 545 present-day Umbrians (with 198 entire mitogenomes) and 28 pre-Roman individuals (obtaining 19 ancient mtDNAs) excavated from the necropolis of Plestia. We found a rather homogeneous distribution of western Eurasian lineages across the region, with few notable exceptions. Contemporary inhabitants of the eastern part, delimited by the Tiber River and the Apennine Mountains, manifest a peculiar mitochondrial proximity to central-eastern Europeans, mainly due to haplogroups U4 and U5a, and an overrepresentation of J (30%) similar to the pre-Roman remains, also excavated in East Umbria. Local genetic continuities are further attested to by six terminal branches (H1e1, J1c3, J2b1, U2e2a, U8b1b1 and K1a4a) shared between ancient and modern mitogenomes. Eventually, we identified multiple inputs from various population sources that likely shaped the mitochondrial gene pool of ancient Umbri over time, since early Neolithic, including gene flows with central-eastern Europe. This diachronic mtDNA portrait of Umbria fits well with the genome-wide population structure identified on the entire peninsula and with historical sources that list the Umbri among the most ancient Italic populations.

The lot-to-lot variability in the mitochondrial genome of controls.

Current research in the biomedical field has illustrated how cell lines used as reference standards can change over time and, more importantly, can affect research and diagnostic results obtained from these cell lines. With the use of increasingly sensitive and highly resolving technologies (e.g., massively parallel sequencing), forensic scientists must be aware of and account for potential variability in the cell lines used as controls in their validation studies and day-to-day casework. In this study, multiple lot numbers from four commonly-used control cell line DNAs were sequenced with massively parallel sequencing on the Ion S5. The variability among these different lots was evaluated, and the effect on forensic laboratory work discussed.

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.

Mitochondrial DNA analysis of a Viking age mass grave in Sweden.

In 1998, a Viking Age mass grave was discovered and excavated at St. Laurence´s churchyard in Sigtuna, Sweden. The excavated bones underwent osteoarchaeological analysis and were assigned to at least 19 individuals. Eleven skeletons showed sharp force trauma from bladed weapons. Mass graves are an unusual finding from this time period, making the burial context extraordinary. To investigate a possible maternal kinship among the individuals, bones and teeth from the skeletal remains were selected for mitochondrial DNA (mtDNA) analysis. Sanger sequencing of short stretches of the hypervariable segments I and II (HVS-I and HVS-II) was performed. A subset of the samples was also analysed by massively parallel sequencing analysis (MPS) of the entire mtDNA genome using the Precision ID mtDNA Whole Genome Panel. A total of 15 unique and three shared mtDNA profiles were obtained. Based on a combination of genetic and archaeological data, we conclude that a minimum of 20 individuals was buried in the mass grave. The majority of the individuals were not maternally related. However, two possible pairs of siblings or mother-child relationships were identified. All individuals were assigned to West Eurasian haplogroups, with a predominance of haplogroup H. Although the remains showed an advanced level of DNA degradation, the combined use of Sanger sequencing and MPS with the Precision ID mtDNA Whole Genome Panel revealed at least partial mtDNA data for all samples.

SD quants-Sensitive detection tetraplex-system for nuclear and mitochondrial DNA quantification and degradation inference.

Measuring the quantity of DNA present in a forensic sample is relevant in a number of ways. First, it informs the analyst about the general DNA content to adjust the volume of DNA extract used for the genotyping assay to the optimal conditions (when possible). Second, quantification values can serve as plausibility checks for the performance of the DNA extraction method used as extraction positive and negative controls demand expected values. Third and relevant to highly compromised specimens, DNA quantification can inform about the degradation state of the DNA extracted from the unknown biological sample and aid the choice of downstream genotyping assays. While there are different, commercial products for the quantification of nuclear DNA available, commercial mitochondrial DNA (mtDNA) quantification systems are rare. Even more so, the simultaneous quantification of nuclear and mtDNA that is of relevance in highly degraded forensic specimens has rarely been described. We present here a novel real-time qPCR based tetraplex system termed SD quants that targets two different-sized mtDNA and a nuclear DNA region and includes an internal positive control to monitor potential inhibition. SD quants was compared to other existing quantification systems and subjected to analysis of severely degraded DNA present in ancient DNA and aged forensic specimens. This study complies with the MIQE (Bustin et al., 2009) guidelines (when applicable).

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 the precision ID whole MtDNA genome panel for forensic analyses.

Mitochondrial DNA (mtDNA) amplification and Massively Parallel Sequencing (MPS) using an early access version of the Precision ID Whole MtDNA Genome Panel (Thermo Fisher Scientific) and the Ion Personal Genome Machine (PGM) were evaluated using 15 forensically relevant samples. Samples were selected to represent typical forensic specimens for mtDNA analysis including hairs, hair shafts, swabs and ancient solid tissue samples (bones and teeth) that were stored in the freezer for up to several years after having been typed with conventional Sanger-type Sequencing and Capillary Electrophoresis. The MPS haplotypes confirmed the earlier results in all samples and provided additional sequence information that improved discrimination power and haplogroup estimation. The results raised the appetite for further experiments to validate and apply the new technology in forensic practice.

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin.

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories.

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.

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.

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.

Full mtGenome reference data: development and characterization of 588 forensic-quality haplotypes representing three U.S. populations.

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

Evidence for frequent and tissue-specific sequence heteroplasmy in human mitochondrial DNA.

Mitochondrial point heteroplasmy is a common event observed not only in patients with mitochondrial diseases but also in healthy individuals. We here report a comprehensive investigation of heteroplasmy occurrence in human including the whole mitochondrial control region from nine different tissue types of 100 individuals. Sanger sequencing was used as a standard method and results were supported by cloning, minisequencing, and massively parallel sequencing. Only 12% of all individuals showed no heteroplasmy, whereas 88% showed at least one heteroplasmic position within the investigated tissues. In 66% of individuals up to 8 positions were affected. The highest relative number of heteroplasmies was detected in muscle and liver (79%, 69%), followed by brain, hair, and heart (36.7%-30.2%). Lower percentages were observed in bone, blood, lung, and buccal cells (19.8%-16.2%). Accumulation of position-specific heteroplasmies was found in muscle (positions 64, 72, 73, 189, and 408), liver (position 72) and brain (partial deletion at position 71). Deeper analysis of these specific positions in muscle revealed a non-random appearance and position-specific dependency on age. MtDNA heteroplasmy frequency and its potential functional importance have been underestimated in the past and its occurrence is ubiquitous and dependent at least on age, tissue, and position-specific mutation rates.

Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens.

Forensic mitochondrial DNA (mtDNA) testing requires appropriate, high quality reference population data for estimating the rarity of questioned haplotypes and, in turn, the strength of the mtDNA evidence. Available reference databases (SWGDAM, EMPOP) currently include information from the mtDNA control region; however, novel methods that quickly and easily recover mtDNA coding region data are becoming increasingly available. Though these assays promise to both facilitate the acquisition of mitochondrial genome (mtGenome) data and maximize the general utility of mtDNA testing in forensics, the appropriate reference data and database tools required for their routine application in forensic casework are lacking. To address this deficiency, we have undertaken an effort to: (1) increase the large-scale availability of high-quality entire mtGenome reference population data, and (2) improve the information technology infrastructure required to access/search mtGenome data and employ them in forensic casework. Here, we describe the application of a data generation and analysis workflow to the development of more than 400 complete, forensic-quality mtGenomes from low DNA quantity blood serum specimens as part of a U.S. National Institute of Justice funded reference population databasing initiative. We discuss the minor modifications made to a published mtGenome Sanger sequencing protocol to maintain a high rate of throughput while minimizing manual reprocessing with these low template samples. The successful use of this semi-automated strategy on forensic-like samples provides practical insight into the feasibility of producing complete mtGenome data in a routine casework environment, and demonstrates that large (>2kb) mtDNA fragments can regularly be recovered from high quality but very low DNA quantity specimens. Further, the detailed empirical data we provide on the amplification success rates across a range of DNA input quantities will be useful moving forward as PCR-based strategies for mtDNA enrichment are considered for targeted next-generation sequencing workflows.

WITHDRAWN: Erratum to "Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM)" [Forensic Sci. Int.: Genet. 7 (2013) 543-549].

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.fsigen.2013.09.007. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Reprint of: Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM).

Insights into the human mitochondrial phylogeny have been primarily achieved by sequencing full mitochondrial genomes (mtGenomes). In forensic genetics (partial) mtGenome information can be used to assign haplotypes to their phylogenetic backgrounds, which may, in turn, have characteristic geographic distributions that would offer useful information in a forensic case. In addition and perhaps even more relevant in the forensic context, haplogroup-specific patterns of mutations form the basis for quality control of mtDNA sequences. The current method for establishing (partial) mtDNA haplotypes is Sanger-type sequencing (STS), which is laborious, time-consuming, and expensive. With the emergence of Next Generation Sequencing (NGS) technologies, the body of available mtDNA data can potentially be extended much more quickly and cost-efficiently. Customized chemistries, laboratory workflows and data analysis packages could support the community and increase the utility of mtDNA analysis in forensics. We have evaluated the performance of mtGenome sequencing using the Personal Genome Machine (PGM) and compared the resulting haplotypes directly with conventional Sanger-type sequencing. A total of 64mtGenomes (>1 million bases) were established that yielded high concordance with the corresponding STS haplotypes (<0.02% differences). About two-thirds of the differences were observed in or around homopolymeric sequence stretches. In addition, the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of alignment software would be desirable to facilitate the application of NGS in mtDNA forensic genetics.

Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM).

Insights into the human mitochondrial phylogeny have been primarily achieved by sequencing full mitochondrial genomes (mtGenomes). In forensic genetics (partial) mtGenome information can be used to assign haplotypes to their phylogenetic backgrounds, which may, in turn, have characteristic geographic distributions that would offer useful information in a forensic case. In addition and perhaps even more relevant in the forensic context, haplogroup-specific patterns of mutations form the basis for quality control of mtDNA sequences. The current method for establishing (partial) mtDNA haplotypes is Sanger-type sequencing (STS), which is laborious, time-consuming, and expensive. With the emergence of Next Generation Sequencing (NGS) technologies, the body of available mtDNA data can potentially be extended much more quickly and cost-efficiently. Customized chemistries, laboratory workflows and data analysis packages could support the community and increase the utility of mtDNA analysis in forensics. We have evaluated the performance of mtGenome sequencing using the Personal Genome Machine (PGM) and compared the resulting haplotypes directly with conventional Sanger-type sequencing. A total of 64mtGenomes (>1 million bases) were established that yielded high concordance with the corresponding STS haplotypes (<0.02% differences). About two-thirds of the differences were observed in or around homopolymeric sequence stretches. In addition, the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of alignment software would be desirable to facilitate the application of NGS in mtDNA forensic genetics.