Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks.

BACKGROUND: DNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity. RESULTS: The protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging. CONCLUSIONS: We propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.

The last flight of F/O Tadeusz Stabrowski. Identification of the polish pilot.

The paper presents the process of identifying an unnamed soldier of the Polish armed forces in the west, whose remains were found in a nameless grave at the municipal cemetery in Le Crotoy in France. The Polish Genetic Database of Victims of Totalitarianism team carried out the research in cooperation with the Ministry of Culture and National Heritage. A comprehensive analysis of autosomal and Y-STR markers was performed. Historical, anthropological, and forensic examinations of the remains were also carried out. The items found with the remains were also examined. Identification based on DNA analysis made it possible to restore the identity of the Polish pilot who died on 11 March 1943 near the French coast, F/O Tadeusz Stabrowski. The airman regained his name in 2018, he was about 26 years old at the time of his death and left behind a grieving wife and son in the United Kingdom. The success of identifying the NN remains was guaranteed by the appointment of an interdisciplinary team consisting of specialists in archaeology, anthropology, history, forensic medicine and forensic genetics. The analysis of historical sources allowed to determine 4 missing airmen whose remains could have been buried in the cemetery in Le Crotoy. An interesting aspect of the research was the cooperation with history enthusiasts and fans of Polish aviation, thanks to which it was finally possible to narrow down the group of pilots sought and reach the family of Tadeusz Stabrowski, who submitted comparative material for research. This is the first case of establishing the identity of a Polish pilot killed in France. Many institutions have been involved in the project, including Polish Ministry of Culture and National Heritage (MDiKN), which partially funded the research.

Skeletal evidence of the ethnic cleansing actions in the Free City of Danzig (1939-1942) based on the KL Stutthof victims analysis.

In the early days of World War II, many of the prominent and influential people of Polish nationality from the Free City of Danzig were arrested by the Germans and sent to the nearby concentration camp KL Stutthof. Nearly a hundred of them died within the next seven months upon their arrival, and were buried in a clandestine mass grave in a nearby forest. However, the exact nature of their death is unknown, as it is unclear what the attitude of the aggressors was toward the victims. We do not know whether there was only one executioner or there were several assassins, nor if the killing methodology was consistent with the other state-regulated executions. The studied material represents the commingled remains of a minimum thirty-four people, possibly all male, aged from under eighteen to over sixty at the time of death. Perimortem traumatic lesions are shown mainly on the skull bones. We asked whether the perimortem trauma lesions visible on the victims' skeletons could be informative on the cause and manner of their death. Our results show the prevalence of the perimortem trauma inflicted by a blunt object are on the parietal bones above the Hat Brim Line (HBL), which is commonly associated with a violent attack. The gunshot trauma was usually localized on the occipital bone or posterior parietal, which could indicate a shot to the back of the head, and this was commonly encountered during executions. No signs of defensive injuries can be explained either by restraining of the hands or by a surprise attack. The abundance and variability of the trauma type can be evident of multiple assailants. Moreover, the multiple impact points detected on several crania prove unnecessary overkill and brutality, which reflects the personal attitudes of the executioners towards the victims.

Black Devils in Normandy-Identification of an Unknown Soldier Found in the Polish War Cemetery of Urville-Langannerie (France).

A paper dedicated to the identification of a Polish soldier from the 1st Armoured Division under the command of General Stanislaw Maczek, who fell in 1944 in Normandy, during World War II. The remains were found at the Urville-Langannerie Polish War Cemetery. A team from the Department of Forensic Genetics at the Pomeranian Medical University in Szczecin, commissioned by the Ministry of Culture Heritage and Sport, exhumed the remains in order to carry out genetic identification tests. A comprehensive anthropological analysis of the heavily degraded remains was carried out, and biological samples were secured for genetic testing. The identification of Jan Dusza is the first case of restoring the identity of an active combatant from the First Armoured Division. In the case analysis, the analysis of mitochondrial DNA in highly degraded biological material proved crucial. Genetic studies decided to reject the original historical hypothesis No. I at their preliminary stage. Regarding hypothesis No. II, a comprehensive genetic analysis of mitochondrial and autosomal DNA was carried out. Comparative material was obtained from the alleged victim's sister. Thanks to the analysis of kinship in the maternal line based on the mtDNA haplotype, it was possible to establish that the remains belong to Jan Dusza, who served in the Podhale Rifle Battalion, part of the Polish 1st Armoured Division. The research was co-financed by the Polish Ministry of Heritage and National Culture.

SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology-Challenges, Protocol Optimization and Success Rates for Different Strains.

The COVID-19 pandemic demonstrated how rapidly various molecular methods can be adapted for a Public Health Emergency. Whether a need arises for whole-genome studies (next-generation sequencing), fast and high-throughput diagnostics (reverse-transcription real-time PCR) or global immunization (construction of mRNA or viral vector vaccines), the scientific community has been able to answer all these calls. In this study, we aimed at the assessment of effectiveness of the commercially available solution for full-genome SARS-CoV-2 sequencing (AmpliSeq™ SARS-CoV-2 Research Panel and Ion AmpliSeq™ Library Kit Plus, Thermo Fisher Scientific). The study is based on 634 samples obtained from patients from Poland, with varying viral load, assigned to a number of lineages. Here, we also present the results of protocol modifications implemented to obtain high-quality genomic data. We found that a modified library preparation protocol required less viral RNA input in order to obtain the optimal library quantity. Concurrently, neither concentration of cDNA nor reamplification of libraries from low-template samples improved the results of sequencing. On the basis of the amplicon success rates, we propose one amplicon to be redesigned, namely, the r1_1.15.1421280, for which less than 50 reads were produced by 44% of samples. Additionally, we found several mutations within different SARS-CoV-2 lineages that cause the neighboring amplicons to underperform. Therefore, due to constant SARS-CoV-2 evolution, we support the idea of conducting ongoing sequence-based surveillance studies to continuously validate commercially available RT-PCR and whole-genome sequencing solutions.

Genetic and phylogeographic evidence for Jewish Holocaust victims at the Sobibór death camp.

Six million Jews were killed by Nazi Germany and its collaborators during World War II. Archaeological excavations in the area of the death camp in Sobibór, Poland, revealed ten sets of human skeletal remains presumptively assigned to Polish victims of the totalitarian regimes. However, their genetic analyses indicate that the remains are of Ashkenazi Jews murdered as part of the mass extermination of European Jews by the Nazi regime and not of otherwise hypothesised non-Jewish partisan combatants. In accordance with traditional Jewish rite, the remains were reburied in the presence of a Rabbi at the place of their discovery.

Molecular Evolution and Epidemiological Characteristics of SARS COV-2 in (Northwestern) Poland.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolved into a worldwide outbreak, with the first Polish cases in February/March 2020. This study aimed to investigate the molecular epidemiology of the circulating virus lineages between March 2020 and February 2021. We performed variant identification, spike mutation pattern analysis, and phylogenetic and evolutionary analyses for 1106 high-coverage whole-genome sequences, implementing maximum likelihood, multiple continuous-time Markov chain, and Bayesian birth-death skyline models. For time trends, logistic regression was used. In the dataset, virus B.1.221 lineage was predominant (15.37%), followed by B.1.258 (15.01%) and B.1.1.29 (11.48%) strains. Three clades were identified, being responsible for 74.41% of infections over the analyzed period. Expansion in variant diversity was observed since September 2020 with increasing frequency of the number in spike substitutions, mainly H69V70 deletion, P681H, N439K, and S98F. In population dynamics inferences, three periods with exponential increase in infection were observed, beginning in March, July, and September 2020, respectively, and were driven by different virus clades. Additionally, a notable increase in infections caused by the B.1.1.7 lineage since February 2021 was noted. Over time, the virus accumulated mutations related to optimized transmissibility; therefore, faster dissemination is reflected by the second wave of epidemics in Poland.

Comparing Genetic Variation among Latin American Immigrants: Implications for Forensic Casework in the Arizona- and Texas-Mexico Borderlands.

The humanitarian crisis on the US-Mexico border is a long-standing and evolving crisis in which nearly 8,000 deaths have been reported in the last two decades. These deaths are largely distributed across the Arizona-Mexico and Texas-Mexico border regions, where demographic trends for immigrants attempting to cross into the United States have shifted dramatically. The demographic change and volume of immigrants seeking shelter in the United States present new challenges for the forensic practitioners entrusted with the identification of individuals who lose their lives during the final segment of their journey. Within this border context, this study investigated how genetic variation inferred from forensically significant microsatellites can provide valuable information on regions of origin for unidentified remains at the group level. To explore how to mobilize these genetic data to inform identification strategies, the authors conducted a comparative genetic analysis of identified and unidentified immigrant cases from the Arizona- and Texas-Mexico contexts, as well as 27 other Latin American groups. Allele frequencies were utilized to calculate FST, and relationships were visually depicted in a multidimensional scaling plot. A Spearman correlation coefficient analysis assessed the strength and significance of population relationships, and an agglomerative clustering analysis assessed population clusters. Results indicate that Arizona-Mexico immigrants have the strongest relationship (>80%) with groups from El Salvador, Guatemala, Mexico, and an indigenous group from southern Mexico. Texas-Mexico immigrants have the strongest relationships (>80%) with groups from Belize, Colombia, Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua. These findings agree with, and are discussed in comparison with, previously reported demographic trends, population genetics research, and population history analyses. The authors emphasize the utility and necessity of coupling genetic variation research with a nuanced anthropological perspective for identification processes in the US-Mexico border context.

Y-Chromosome Genetic Analysis of Modern Polish Population.

The study presents a full analysis of the Y-chromosome variability of the modern male Polish population. It is the first study of the Polish population to be conducted with such a large set of data (2,705 individuals), which includes genetic information from inhabitants of all voivodeships, i.e., the first administrative level, in the country and the vast majority of its counties, i.e., the second level. In addition, the available data were divided into clusters corresponding to more natural geographic regions. Genetic analysis included the estimation of F ST distances, the visualization with the use of multidimensional scaling plots and analysis of molecular variance. Y-chromosome binary haplogroups were classified and visualized with the use of interpolation maps. Results showed that the level of differentiation within Polish population is quite low, but some differences were indicated. It was confirmed that the Polish population is characterized by a high degree of homogeneity, with only slight genetic differences being observed at the regional level. The use of regional clustering as an alternative to counties and voivodeships provided a more detailed view of the genetic structure of the population. Those regional differences identified in the present study highlighted the need for additional division of the population by cultural and ethnic criteria in such studies rather than just by geographical or administrative regionalization.

The challenge of predicting human pigmentation traits in degraded bone samples with the MPS-based HIrisPlex-S system.

Identification of human remains is an important part of human DNA analysis studies. STR and mitochondrial DNA markers are well suited for the analysis of degraded biological samples including bone material. However, these DNA markers may be useless when reference material is not available. In these cases, predictive DNA analysis can support the process of human identification by providing investigative leads. Forensic DNA phenotyping has progressed significantly by offering new methods based on massively parallel sequencing technology, but the frequent degradation processes observed in skeletal remains can make analysis of such samples challenging. In this study, we demonstrate the usefulness of a recently established Ion AmpliSeqTM HIrisPlex-S panel using Ion Torrent technology for analyzing bone samples that show different levels of DNA degradation. In total, 63 bone samples at post-mortem intervals up to almost 80 years were genotyped and eye, hair and skin colour predictions were performed using the HIrisPlex-S models. Following the recommended coverage thresholds, it was possible to establish full DNA profiles comprising of 41 DNA variants for 35 samples (55.6%). For 5 samples (7.9%) no DNA profiles were generated. The remaining 23 samples (36.5%) produced partial profiles and showed a clear underperformance of 3 HIrisPlex-S SNPs - rs1545397 (OCA2), rs1470608 (OCA2) and rs10756819 (BNC2), all used for skin colour prediction only. None of the 23 samples gave complete genotypes needed for skin colour prediction was obtained, and in 7 of them (25.9%) the 3 underperformed SNPs were the cause. At the same time, the prediction of eye and hair colour using complete IrisPlex and HIrisPlex profiles could be made for these 23 samples in 20 (87.0%) and 12 cases (52.2%), respectively. Complete HIrisPlex-S profiles were generated from as little as 49 pg of template DNA. Five samples for which the HIrisPlex-S analysis failed, consistently failed in standard STR analysis. Importantly, the 3 underperforming SNPs produced significantly lower number of reads in good quality samples. Nonetheless, the AUC loss resulting from missing data for these 3 SNPs is not considered large (≤0.004) and the prediction of pigmentation from partial profiles is also available in the current HPS tool. The study shows that DNA degradation and the resulting loss of data are the most serious challenge to DNA phenotyping of skeletal remains. Although the newly developed HIrisPlex-S panel has been successfully validated in the current research, primer redesign for the 3 underperforming SNPs in the MPS design should be considered in the future.

Is MPS always the answer? Use of two PCR-based methods for Y-chromosomal haplotyping in highly and moderately degraded bone material.

Forensic and population genetics often rely on Y-chromosomal studies. Whether it is a human identification case, trace evidence examination or phylogenetic analysis, a Y-STR haplotype is an important tool in the hands of law enforcement agencies. A common obstacle in achieving satisfactory results in all of the above mentioned circumstances, is low DNA quantity and quality within samples obtained. In this study we have examined Y-STR haplotypes in 75 bone material samples, coming from different time periods. For this purpose we have chosen YFiler Plus PCR Amplification Kit (ThermoFisher Scientific) and ForenSeq Signature DNA Prep Kit (Verogen Inc.), which use two different allele calling technologies - capillary electrophoresis and Massively Parallel Sequencing respectively. Full profiles were obtained from DNA extracts with as little as 0.1896 ng (Degradation Index 1.3) (ForenSeq) and 0.0591 ng (Degradation Index 26.8) (YFiler Plus) DNA input. The results that we present in this paper show differences in amplification rates between common markers in both kits. The differences strictly reflect mean amplicon length of markers. This, however, does not seem to influence Y-haplogroup estimation results noticeably. In one sample a discordance occurred between haplotypes obtained with both methods, where a 24 allele was called in DYS390 marker by capillary electrophoresis, while for the same sample in this locus a 23 allele was shown with MPS. A reason for this is yet to be investigated. The sequence analysis revealed a significant variation between isometric alleles, especially within repetitive regions of studied Y-STR markers.

Analysis of male specific region of the human Y chromosome sheds light on historical events in Nazi occupied eastern Poland.

In Poland, during the World War II, almost 3 million people were killed during the Nazi occupation, and about 570,000 during the Soviet occupation. Furthermore, historians have estimated that after the World War II at least 30,000 people were killed during the Stalinist regime in Poland (1944-1956). The exact number is unknown, because both executions and burials were kept secret. Thousands of people just vanished. As a response to those events, forensic scientists from the Pomeranian Medical University in Szczecin in cooperation with historians from the Institute of National Remembrance started the project of the Polish Genetic Database of Victims of Totalitarianism, which aim is to identify victims killed in the years 1939-1956. Several exhumations were done under the project, with the biggest one done in Bialystok. According to the information gathered by local historians, a detention centre in Bialystok was the place of the secret burials in late 1940s and 1950s. Surprisingly, except few graves from the post-war period, most of the burials found in Bialystok indicated that majority the victims were probably local civilians who died during the Nazi occupation. Unfortunately, data concerning what happened in the detention ward during that period of time is not very detailed. What was known is that people who got incarcerated were "political prisoners" what, according to Nazi politics, was based on their nationality, religion and activity against the Third Reich. The aim of this research was to test genetically the remains found in Bialystok to determine their possible ethnic background, in order to shed new light on the victims and what happened in the Bialystok detention centre during the Nazi occupation. The analysis of male specific region of the human Y chromosome shows that including phylogenetic analysis into the complex process led by the Polish Genetic Database of Victims of Totalitarianism may help with the final identification of hundreds of anonymous victims.

In search for the grave of 100 Poles executed on March 20, 1942 in Zgierz, Poland - research by SIGO (Network for Genetic Identification of Victims).

It can be reasonably assumed that remains exhumed in 2012 and 2013 during archaeological explorations conducted in the Lucmierz Forest, an important area on the map of the German Nazi terror in the region of Lodz (Poland), are in fact the remains of a hundred Poles murdered by the Nazis in Zgierz on March 20, 1942. By virtue of a decision of the Polish Institute of National Remembrance's Commission for the Prosecution of Crimes Against the Polish Nation, the verification of this research hypothesis was entrusted to SIGO (Network for Genetic Identification of Victims) Consortium appointed by virtue of an agreement of December 11, 2015. The Consortium is an extension of the PBGOT (Polish Genetic Database of Totalitarianisms Victims). So far, the researchers have retrieved 14 DNA profiles from among the examined remains, including 12 male and 2 female profiles. Furthermore, 12 DNA profiles of the victims' family members have been collected. Due to the fact that next-of-kin relatives of the victims of the Zgierz massacre are of advanced age, it is of key importance to collect genetic material as soon as possible from the other surviving family members, identified on the basis of a list of victims that has been nearly completely compiled by the Polish Institute of National Remembrance (IPN) and is presented in this paper.

A case study of an unknown mass grave - Hostages killed 70 years ago by a Nazi firing squad identified thanks to genetics.

Almost 6 million people died in Poland during the Nazi occupation and about 570 thousand during the Soviet occupation. But the end of the war was not the end of the trauma. Historians estimate that at least 30 thousand people were killed during the Stalinist regime in Poland. In 2012 the Institute of National Remembrance started to search for hidden burials of victims of communism. Many exhumations were carried out under the project. One of them took place in Bialystok, eastern Poland. According to information gathered by local historians, a detention centre in the heart of city was the place of secret burials of victims of the communist regime. During the exhumation work a burial pit with the remains of 24 victims was found. It's characteristics supported the hypothesis that these people were shot on the spot, in a mass execution during the Nazi occupation. Historians knew of only one such execution, but its victims - according to the available records - were supposed to have been exhumed at the end of the war. Exhumation works and the discovery of the discussed mass grave put in question the events of 1944, which would have been impossible without the field work. The first identifications confirmed the doubts of historians, since both the results of genetic profiling and the conducted anthropological analysis revealed that at the end of the war a mistake was made, and bodies other than those suspected had been exhumed. Having established this fact, the mass grave created at that time should be investigated to reveal the identity of the remains uncovered then.