Identification of skeletal remains in Croatia and Bosnia and Herzegovina, including the homeland war - a 30-year review.

Over the past 30 years, forensic experts from Croatia and Bosnia and Herzegovina have embraced advanced technologies and innovations to enable great efficacy and proficiency in the identification of war victims. The wartime events in the countries of former Yugoslavia greatly influenced the application of the selected DNA analyses as routine tools for the identification of skeletal remains, especially those from mass graves. Initially, the work was challenging because of the magnitude of the events, technical aspects, and political aspects. Collaboration with reputable foreign forensic experts helped tremendously in the efforts to start applying DNA analysis routinely and with increasing success. In this article, we reviewed the most significant achievements related to the application of DNA analysis in identifying skeletal remains in situations where standard identification methods were insufficient.

A New Tool for Probabilistic Assessment of MPS Data Associated with mtDNA Mixtures.

Mitochondrial (mt) DNA plays an important role in the fields of forensic and clinical genetics, molecular anthropology, and population genetics, with mixture interpretation being of particular interest in medical and forensic genetics. The high copy number, haploid state (only a single haplotype contributed per individual), high mutation rate, and well-known phylogeny of mtDNA, makes it an attractive marker for mixture deconvolution in damaged and low quantity samples of all types. Given the desire to deconvolute mtDNA mixtures, the goals of this study were to (1) create a new software, MixtureAceMT™, to deconvolute mtDNA mixtures by assessing and combining two existing software tools, MixtureAce™ and Mixemt, (2) create a dataset of in-silico MPS mixtures from whole mitogenome haplotypes representing a diverse set of population groups, and consisting of two and three contributors at different dilution ratios, and (3) since amplicon targeted sequencing is desirable, and is a commonly used approach in forensic laboratories, create biological mixture data associated with two amplification kits: PowerSeq™ Whole Genome Mito (Promega™, Madison, WI, USA) and Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by AB™, Waltham, MA, USA) to further validate the software for use in forensic laboratories. MixtureAceMT™ provides a user-friendly interface while reducing confounding features such as NUMTs and noise, reducing traditionally prohibitive processing times. The new software was able to detect the correct contributing haplogroups and closely estimate contributor proportions in sequencing data generated from small amplicons for mixtures with minor contributions of ≥5%. A challenge of mixture deconvolution using small amplicon sequencing is the potential generation of spurious haplogroups resulting from private mutations that differ from Phylotree. MixtureAceMT™ was able to resolve these additional haplogroups by including known haplotype/s in the evaluation. In addition, for some samples, the inclusion of known haplotypes was also able to resolve trace contributors (minor contribution 1-2%), which remain challenging to resolve even with deep sequencing.

Massively Parallel Sequencing of the Mitogenome from Human Hair Shafts in Forensic Investigations.

This article highlights methods used to perform DNA extraction, mitochondrial DNA quantification, multiplex PCR amplification, amplicon-based massively parallel sequencing, and data analysis of the mitochondrial genome (mitogenome) from human hair shafts. The focus is on applications to forensic casework, but this set of protocols can be used for any purpose involving small cuttings (as small as 1 to 5 mm) of human hair shafts up to 40 years from the time of collection. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of mitochondrial DNA from human hair shafts Basic Protocol 2: Quantification of mitochondrial DNA (copies/µl) Basic Protocol 3: Multiplex amplification of the mitogenome Basic Protocol 4: Library preparation and sequencing of mitogenome amplicons Basic Protocol 5: Data analysis of mitogenome haplotypes.

Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair.

While hair shafts are a common evidence type in forensic cases, they are often excluded from DNA analysis due to their limited DNA quantity and quality. Mitochondrial (mt) DNA sequencing is the method of choice when working with rootless hair shaft fragments due to the elevated copy number of mtDNA and the highly degraded nature of nuclear (n) DNA. Using massively parallel sequencing (MPS) of the mitochondrial (mito) genome, we studied the impact of hair age (time since collection) and physical characteristics (hair diameter, medullary structure, and length of hair tested) on mtDNA recovery and MPS data quality. Hair shaft cuttings of 1 and 5 mm from hairs less than five years to 46 years of age from 60 donors were characterized microscopically. Mitogenome sequences were generated using the Promega PowerSeqTM Whole Mito System prototype kit and the Illumina MiSeq instrument. Reportable mitogenome sequences were obtained from all hairs up to 27 years of age (37 donors), with at least 98% of the mitogenome reported for more than 94% of the 74 hair samples analyzed; the minimum reported sequence was 88%. Furthermore, data from the 1 and 5 mm replicates gave concordant haplotypes. As expected, mtDNA yield decreased, mtDNA degradation increased, and mitogenome MPS data quality declined as the age of the hair increased. Hair diameter and medullary structure had minimal impact on yield and data quality. Our findings support that MPS is a robust and reliable method for routinely generating mitogenome sequences from 1 and 5 mm hair shaft samples up to 27 years of age, which is of interest to the forensic community, biological anthropologists, and medical geneticists.

Exploring statistical weight estimates for mitochondrial DNA matches involving heteroplasmy.

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows forensic laboratories to report heteroplasmy on a routine basis. Statistical approaches will be needed to determine the relative frequency of observing an mtDNA haplotype when including the presence of a heteroplasmic site. Here, we examined 1301 control region (CR) sequences, collected from individuals in four major population groups (European, African, Asian, and Latino), and covering 24 geographically distributed haplogroups, to assess the rates of point heteroplasmy (PHP) on an individual and nucleotide position (np) basis. With a minor allele frequency (MAF) threshold of 2%, the data was similar across population groups, with an overall PHP rate of 37.7%, and the majority of heteroplasmic individuals (77.3%) having only one site of heteroplasmy. The majority (75.2%) of identified PHPs had an MAF of 2-10%, and were observed at 12.6% of the nps across the CR. Both the broad and phylogenetic testing suggested that in many cases the low number of observations of heteroplasmy at any one np results in a lack of statistical association. The posterior frequency estimates, which skew conservative to a degree depending on the sample size in a given haplogroup, had a mean of 0.152 (SD 0.134) and ranged from 0.031 to 0.83. As expected, posterior frequency estimates decreased in accordance with 1/n as the sample size (n) increased. This provides a proposed conservative statistical framework for assessing haplotype/heteroplasmy matches when applying an MPS technique in forensic cases and will allow for continual refinement as more data is generated, both within the CR and across the mitochondrial genome.

MaSTR™: an effective probabilistic genotyping tool for interpretation of STR mixtures associated with differentially degraded DNA.

The recently developed probabilistic genotyping software package MaSTR™ (SoftGenetics LLC) was used to develop statistical weight estimates for a variety of two-person STR mixture profiles with differentially degraded sources of DNA. A total of 864 analyses, on 144 two-person profiles, were performed. Mixture ratios ranged from 1:1 to 1:10, including pristine sources of DNA and various combinations of artificially degraded DNA (average size fragments of 150 or 250 bps). Quantities of DNA template were varied (0.1 to 0.5 ngs of total input) and MaSTR™ analysis was performed with eight chains of 10,000 or 40,000 iterations, with or without a conditioning profile to generate likelihood ratio (LR) values. Overall, the software performed as expected. The resulting log(LR) values for pristine mixture profiles were typically greater than 1030. Lower-quality mixture data associated with sources of DNA at ~ 0.05 ngs for each contributor resulted in peak imbalance and allelic dropout which reduced the weight in support of a contributor. This was exacerbated by higher levels of degradation, with some instances resulting in log(LR) values in support of an exclusion. These studies provide additional support for the use of probabilistic genotyping software solutions in forensic investigations, addressing concerns raised by the President's Council of Advisors on Science and Technology (PCAST).

Damage patterns observed in mtDNA control region MPS data for a range of template concentrations and when using different amplification approaches.

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows practitioners the ability to fully resolve heteroplasmic sites. In forensic DNA analysis, identifying heteroplasmy (a naturally occurring mixture of two mtDNA profiles) can provide additional mtDNA profile information which can lead to an increase in the discrimination potential of an mtDNA match between an evidentiary sample and reference source. Forensic samples such as hair and skeletal remains, especially older, more compromised samples, can often exhibit DNA damage. Because both damage and heteroplasmy can manifest as a mixture of two nucleotides, it is important to differentiate between the two conditions when interpreting mtDNA MPS data. In this study, DNA damage was applied under controlled conditions to samples containing a range of template concentrations, including some with identified heteroplasmy. Damage was applied via storage in water at room temperature on samples diluted before or after storage to mimic low template scenarios. Damage was assessed with respect to the following areas: mtDNA quantification and degradation ratios, MPS read depth, MPS profile results, overall damage rates, and the interpretation of heteroplasmy. Datasets were generated to assess and compare two different amplification and library preparation strategies: the Promega PowerSeq™ CRM Nested System kit and a 1.16 kb target amplicon of the entire mtDNA control region followed by a Nextera® XT library preparation. The results of this study provide an evaluation of the Promega 10-plex MPS procedure as an improved process to mitigate the impact of mtDNA damage on low template samples. Some of the negative effects of damage observed in this study were a decrease in mtDNA yield by 20-30% and lower quality MPS sequencing results. These effects were observed more frequently when samples were diluted prior to inducing damage, illustrating that low template samples are more susceptible to damage. The findings of this study will assist forensic laboratories in differentiating between damage and heteroplasmy, which is essential when developing robust mtDNA MPS interpretation guidelines such as setting appropriate reporting thresholds.

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulic.

Sister Marija Krucifiksa Kozulic (1852-1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulic (1861-1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains. Thus, mitochondrial genome sequencing of the long bones was performed to sort the remains by mitochondrial haplotype. Two similar but unique haplotypes belonging to haplogroup H1bu were identified, and samples from these bones were subjected to autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing. Although only partial profiles were obtained, the data were sufficient for kinship analysis with the profile of a paternal niece of Sister Marija (Fides Kozulic). The data indicate that it is 574,195-fold more likely that the two sets of skeletal remains represent 2nd-degree relatives of Fides than sisters who are unrelated to Fides. Although it is impossible to discern which set of remains belongs to Marija and which belongs to Tereza, forensic genomics methods have enabled identification of the sisters.

Characterization of background noise in MiSeq MPS data when sequencing human mitochondrial DNA from various sample sources and library preparation methods.

Improved resolution of massively parallel sequencing (MPS) allows for the characterization of mitochondrial (mt) DNA heteroplasmy to levels previously unattainable with traditional sequencing approaches. An essential criterion for the reporting of heteroplasmy is the ability of the MPS method to distinguish minor sequence variants (MSVs) from system noise, or error. Therefore, an assessment of the background noise in the MPS method is desirable to identify the point at which reliable data can be reported. Substitution and sequence specific error (SSE) was evaluated for a variety of sample types and two library preparations. Substitution error rates ranged from 0.18 to 0.49 per 100 nucleotides with C positions generally having the highest rate of misincorporation. Comparison of error rates across sample types indicated a significant increase for samples with damaged DNA. The positions of error were varied across datasets (pairwise concordance 0-68%), but had greater consistency within the damaged samples (80-96%). The most commonly observed motif preceding error in forward reads was CCG, while GGT was most common in reverse reads, both consistent with previous findings. The findings illustrate that for datasets containing samples with damaged DNA, reporting thresholds for heteroplasmy may have to be modified and individual sites with error levels exceeding thresholds should be scrutinized. Collectively, the shifting error profiles observed across the various sample types and library preparation methods demonstrates the need for an assessment of error under these varying circumstances. Characterization of the applicable background noise will help to ensure that thresholds are reliably set for detection of true MSVs.

Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems.

Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.

Recovery of mtDNA from unfired metallic ammunition components with an assessment of sequence profile quality and DNA damage through MPS analysis.

Recovery of suitable amounts of quality DNA from copper and brass surfaces, like those encountered in ammunition, has been a challenge for the forensic community. The ability of copper ions to rapidly facilitate oxidative damage leading to fragmentation of DNA significantly reduces the pool of templates for PCR amplification. We compared two methods for recovering mitochondrial (mt) DNA from the surface of unfired copper projectiles, brass casings, and aluminum casings, and found that using a cotton swab moistened with 0.5M EDTA was the favored approach, especially when the metallic surface was etched. Degradation was significantly higher for DNA samples recovered from copper and brass surfaces, when compared to aluminum. Massively parallel sequencing (MPS) of the control region, using the PowerSeq™ CRM Nested System kit and the Illumina MiSeq instrument, produced full haplotypes for aluminum samples regardless of the method used to deposit or collect DNA, while less than 60% of the copper and brass samples produced partial or full profile information. Touch DNA collected from copper and brass samples produced higher rates of partial or full MPS profile information (∼88-96%), while collection with 0.5M EDTA produced better results than when collection was performed with water; average of ∼70% versus ∼47%. While MPS data was not impacted by noise in the sequencing process, a higher than expected rate of noise was observed, potentially due to an increase in low-level damage lesions. Noise patterns were strikingly different when compared to control data, suggesting that noisy sites may be predictable when testing samples with high levels of oxidative damage. Library preparation was a poor predictor of MPS data quality, as a large percentage of reads did not align with the reference genome. This may impact the number of samples that can be run when a deep-coverage MPS approach is being considered for analysis of mtDNA heteroplasmy. Overall, when applying an MPS approach to the analysis of mtDNA recovered from ammunition, results are expected from touch DNA, will be limited for copper and brass components when the DNA is exposed to an aqueous environment, and DNA degradation will be accelerated when DNA comes in contact with copper or brass surfaces. Practitioners should consider collecting DNA from metallic surfaces with 0.5M EDTA, as this will maximize yield and mitigate degradation. The results of this study directly impact MPS analysis of minor mtDNA sequence variants from metallic surfaces, and are particularly relevant to forensic investigations.

Deep-Coverage MPS Analysis of Heteroplasmic Variants within the mtGenome Allows for Frequent Differentiation of Maternal Relatives.

Abstract: Distinguishing between maternal relatives through mitochondrial (mt) DNA sequence analysis has been a longstanding desire of the forensic community. Using a deep-coverage, massively parallel sequencing (DCMPS) approach, we studied the pattern of mtDNA heteroplasmy across the mtgenomes of 39 mother-child pairs of European decent; haplogroups H, J, K, R, T, U, and X. Both shared and differentiating heteroplasmy were observed on a frequent basis in these closely related maternal relatives, with the minor variant often presented as 2-10% of the sequencing reads. A total of 17 pairs exhibited differentiating heteroplasmy (44%), with the majority of sites (76%, 16 of 21) occurring in the coding region, further illustrating the value of conducting sequence analysis on the entire mtgenome. A number of the sites of differentiating heteroplasmy resulted in non-synonymous changes in protein sequence (5 of 21), and to changes in transfer or ribosomal RNA sequences (5 of 21), highlighting the potentially deleterious nature of these heteroplasmic states. Shared heteroplasmy was observed in 12 of the 39 mother-child pairs (31%), with no duplicate sites of either differentiating or shared heteroplasmy observed; a single nucleotide position (16093) was duplicated between the data sets. Finally, rates of heteroplasmy in blood and buccal cells were compared, as it is known that rates can vary across tissue types, with similar observations in the current study. Our data support the view that differentiating heteroplasmy across the mtgenome can be used to frequently distinguish maternal relatives, and could be of interest to both the medical genetics and forensic communities.

Assessing heteroplasmic variant drift in the mtDNA control region of human hairs using an MPS approach.

Resolution of mitochondrial (mt) DNA heteroplasmy is possible when applying a massively parallel sequencing (MPS) approach. However, interpretation criteria for matching heteroplasmic sequences will need to be established that address a number of important topics, including the drift of variants in sample types such as human hair shafts. Prior to MPS analysis, we compared three different DNA extraction methods for hair using a custom mtDNA quantitative PCR (mtqPCR) assay, and found that a method involving bead capture significantly outperformed methods currently in place in forensic laboratories. The findings were similar for both fine (head) and coarse (pubic) hairs. Using the favored DNA extraction approach, hair shaft extracts were subjected to MPS analysis to assess heteroplasmic drift and the potential impact of the observations on interpretation of mtDNA MPS data. Hairs from different regions of the head were evaluated in individuals with varying percentages of heteroplasmy (low-level, high-level, and no detectable heteroplasmy), as measured in buccal and blood cells. The range of variant ratios was broad and was not significantly different between individuals in the low and high-level groups. While the range was also broad for the group of individuals with no heteroplasmy, the vast majority of hairs from these donors still exhibited a lack of heteroplasmy. A model was developed to predict the amount of heteroplasmy expected in hair samples when knowledge of the percentage of heteroplasmy in buccal cells is available. While significant, the model was best applied when levels of heteroplasmy in buccal cells was high. No correlation was observed between rates of heteroplasmy in blood cells and the predicted amount of heteroplasmy in hairs. Of particular interest, unexpected sites of mixed mtDNA sequence that could be interpreted as heteroplasmy were observed for 13% of the 75 hairs tested. These sites can be explained as heteroplasmy not observed in buccal or blood cells, or sites of DNA damage, with inherent heteroplasmy a likely cause, possibly due to de novo mutation events. Overall, when applying an MPS approach to hair analysis, heteroplasmic variant ratios may be quite different than those observed in blood cells, may be correlated to rates in buccal cells, and may include unexpected mixed sites. The results of this study directly impact MPS analysis of minor sequence variants from hair samples, and are particularly relevant to clinical and forensic investigations.

Evaluation of GeneMarker® HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment.

Existing software has not allowed for effective alignment of mitochondrial (mt) DNA sequence data generated using a massively parallel sequencing (MPS) approach, combined with the ability to perform a detailed assessment of the data. The regions of sequence that are typically difficult to align are homopolymeric stretches, isolated patterns of SNPs (single nucleotide polymorphisms), and INDELs (insertions/deletions). A custom software solution, GeneMarker® HTS, was developed and evaluated to address these limitations, and to provide a user-friendly interface for forensic practitioners and others interested in mtDNA analysis of MPS data. GeneMarker® HTS generates an exportable consensus mtDNA sequence that produces phylogenetically correct SNP and INDEL calls using a customizable motif-based alignment algorithm. Sequence data from 500 individuals, with various alignment asymmetries and levels of heteroplasmy, were used to assess the software. Accuracy in producing mtDNA haplotypes, the ability to correctly identify low-level heteroplasmic sequence variants, and the user-based features of the software were evaluated. Analyzed sequences yielded correct mtDNA haplotypes, and heteroplasmic variants were properly identified with minimal manual interpretation. The software offers numerous user-defined parameters for filtering the data that address the interests of researchers and practitioners, and provides multiple options for viewing and navigating through the data.

MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment.

The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.

Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data.

Resolution of mitochondrial (mt) DNA heteroplasmy is now possible when applying a massively parallel sequencing (MPS) approach, including minor components down to 1%. However, reporting thresholds and interpretation criteria will need to be established for calling heteroplasmic variants that address a number of important topics, one of which is DNA damage. We assessed the impact of increasing amounts of DNA damage on the interpretation of minor component sequence variants in the mtDNA control region, including low-level mixed sites. A passive approach was used to evaluate the impact of storage conditions, and an active approach was employed to accelerate the process of hydrolytic damage (for example, replication errors associated with depurination events). The patterns of damage were compared and assessed in relation to damage typically encountered in poor quality samples. As expected, the number of miscoding lesions increased as conditions worsened. Single nucleotide polymorphisms (SNPs) associated with miscoding lesions were indistinguishable from innate heteroplasmy and were most often observed as 1-2% of the total sequencing reads. Numerous examples of miscoding lesions above 2% were identified, including two complete changes in the nucleotide sequence, presenting a challenge when assessing the placement of reporting thresholds for heteroplasmy. To mitigate the impact, replication of miscoding lesions was not observed in stored samples, and was rarely seen in data associated with accelerated hydrolysis. In addition, a significant decrease in the expected transition:transversion ratio was observed, providing a useful tool for predicting the presence of damage-induced lesions. The results of this study directly impact MPS analysis of minor sequence variants from poorly preserved DNA extracts, and when biological samples have been exposed to agents that induce DNA damage. These findings are particularly relevant to clinical and forensic investigations.

Cultural inter-population differences do not reflect biological distances: an example of interdisciplinary analysis of populations from Eastern Adriatic coast.

AIM: To compare the population group from the Sopot graveyard with population groups from traditional Croatian medieval graveyards by using anthropological, craniometrics, and mitochondrial (mtDNA) analysis and to examine if the cultural differences between population groups reflect biological differences. METHODS: We determined sex, age at death, pathological, and traumatic changes of skeletal remains from the Sopot graveyard and compared them with a cumulative medieval sample from the same region. We also performed principal component analysis to compare skeletal remains from Sopot with those from Ostrovica and other Central European samples according to 8 cranial measurements. Finally, we compared 46 skeletons from Sopot with medieval (Ostrovica) and contemporary populations using mDNA haplogroup profiling. RESULTS: The remains from Sopot were similar to the cumulative sample in lifestyle and quality of life markers. Principal component analysis showed that they were closely related to Eastern Adriatic coast sites (including Ostrovica and Sopot) in terms of cranial morphology, indicating similar biological makeup. According to mDNA testing, Sopot population showed no significant differences in the haplogroup prevalence from either medieval or contemporary populations. CONCLUSION: This study shows that the Sopot population does not significantly differ from other medieval populations from this area. Besides similar quality of life markers, these populations also had similar biological markers. Substantial archeological differences can therefore be attributed to apparent cultural influences, which in this case do not reflect biological differences.

Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA.

The manifestation of mitochondrial DNA (mtDNA) diseases depends on the frequency of heteroplasmy (the presence of several alleles in an individual), yet its transmission across generations cannot be readily predicted owing to a lack of data on the size of the mtDNA bottleneck during oogenesis. For deleterious heteroplasmies, a severe bottleneck may abruptly transform a benign (low) frequency in a mother into a disease-causing (high) frequency in her child. Here we present a high-resolution study of heteroplasmy transmission conducted on blood and buccal mtDNA of 39 healthy mother-child pairs of European ancestry (a total of 156 samples, each sequenced at ∼20,000× per site). On average, each individual carried one heteroplasmy, and one in eight individuals carried a disease-associated heteroplasmy, with minor allele frequency ≥1%. We observed frequent drastic heteroplasmy frequency shifts between generations and estimated the effective size of the germ-line mtDNA bottleneck at only ∼30-35 (interquartile range from 9 to 141). Accounting for heteroplasmies, we estimated the mtDNA germ-line mutation rate at 1.3 × 10(-8) (interquartile range from 4.2 × 10(-9) to 4.1 × 10(-8)) mutations per site per year, an order of magnitude higher than for nuclear DNA. Notably, we found a positive association between the number of heteroplasmies in a child and maternal age at fertilization, likely attributable to oocyte aging. This study also took advantage of droplet digital PCR (ddPCR) to validate heteroplasmies and confirm a de novo mutation. Our results can be used to predict the transmission of disease-causing mtDNA variants and illuminate evolutionary dynamics of the mitochondrial genome.

Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq.

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible. However, in order to bring these approaches into forensic laboratories and subsequently as accepted scientific information in a court of law, validated methods will be required to produce and analyze NGS data. We report here on the development of an optimized approach to NGS analysis for the mtDNA genome (mtgenome) using the Illumina MiSeq instrument. This optimized protocol allows for the production of more than 5 gigabases of mtDNA sequence per run, sufficient for detection and reliable reporting of minor heteroplasmic variants down to approximately 0.5-1.0% when multiplexing twelve samples. Depending on sample throughput needs, sequence coverage rates can be set at various levels, but were optimized here for at least 5000 reads. In addition, analysis parameters are provided for a commercially available software package that identify the highest quality sequencing reads and effectively filter out sequencing-based noise. With this method it will be possible to measure the rates of low-level heteroplasmy across the mtgenome, evaluate the transmission of heteroplasmy between the generations of maternal lineages, and assess the drift of variant sequences between different tissue types within an individual.