Recommendations of the Polish-speaking Working Group of the International Society for Forensic Genetics (ISFG-PL) regarding the disclosure of biological traces and the handling of evidence for identification tests. / Zalecenia Polskojezycznej Grupy Miedzynarodowego Towarzystwa Genetyki Sadowej (ISFG-PL) dotyczace ujawniania i identyfikacji sladów biologicznych przeznaczonych do badan genetycznych.

The purpose of this paper is to formulate recommendations for the disclosure of biological traces in the laboratory and the handling of forensic evidence submitted for identification tests, recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics. The paper organizes the knowledge of the most relevant stages of preliminary analysis of biological traces based on both literature sources and those resulting from years of research practice. Recommendations formulated in the course of multi-stage expert consultations contained in this study should be used in the development of laboratory procedures applied during the execution.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument.

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70-80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone. The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains.

Assessing the utility of quantitative and qualitative metrics in the DNA quantification process of skeletal remains for autosomal and Y-chromosome STR amplification purposes.

In historical cases, ancient DNA investigations and missing persons identification, teeth or bone samples are often the only and almost always the best biological material available for DNA typing. On the other hand, DNA obtained from bone material may be characterized by a high degradation index (DI) or its low content, or DNA tests cannot be repeated due to bone piece size limitation. That is often the effect of the environment in which the material was placed and the time during which exposure to unfavorable environmental factors took place. Therefore, it is very important to use appropriate procedures related to STR analysis. For our study, we selected 80 challenging bone samples. The amount of DNA was compared in qPCR using Quantifiler™ Trio DNA Quantification Kit and Investigator® Quantiplex® Pro RGQ. All qPCR results were confirmed by PCR-CE. The results of DNA concentrations and the assigned degradation index (DI) differed significantly within analyzed samples (~10%). Additionally, the Y-chromosome DI also differed from the autosomal DI in the samples. The difference in degradation indexes could explain the lower Y-chromosome amplification success rate compared to autosomal e.g. during human identification process. The results indicate that performing two DNA quantifications with the use of two different kits (primers sets) allows for a much more precise evaluation of the DNA quality and quantity in the isolate. We suggest that at least one of two suggested DNA concentration measurements should be based on an additional determination of the Y chromosome degradation index. Altogether, it allows for rational isolate management, especially when the volume is limited and the sample is unique.

'Quality of life' concept in Cohesion Policy evaluation in Poland, 2004-2020.

Quality of life (QoL) is a fashionable concept widely used not only in many scientific fields but also in strategic documents, political speeches, and newspaper headlines. Although QoL is a crucial element of political agendas and a goal of public policies, there is little evidence on how this concept has been incorporated in policy analysis and evaluation. This study presents how the QoL has so far been defined, contextualised, and measured in the European Union Cohesion Policy evaluation in Poland, based on the analysis of 1431 evaluation reports for the period 2004-2020. The findings show that the interest in the QoL is growing, and almost one-third of reports refer to the term. However, the concept is usually not clearly defined, and the number of evaluations with actual questions concerning QoL is limited. Moreover, the methodological approach to QoL measurement is rudimentary, limited to surveys of beneficiaries or end-users.

Does evaluation quality matter? Quantitative analysis of the use of evaluation findings in the field of cohesion policy in Poland.

Numerous studies have examined the impact of evaluation quality on the use of evaluation, yet the findings are still inconclusive and only derived from examples in countries with a well-established evaluation culture. This article examines the relationship between evaluation quality and the instrumental use of evaluation in administrations with a low maturity of evaluation culture using Poland as a case study. The research method employs quantitative analysis of datasets regarding evaluation quality and recommendation use from the Polish cohesion policy evaluation system. The use of evaluation findings was measured at two stages: acceptance of recommendations and actual implementation. The results show that the impact of evaluation quality on the use of evaluation is limited and sometimes puzzling. While perceived utility increases the odds of accepting recommendations, the quality of analysis and conclusions reduces it. The actual implementation of recommendations is supported by the robustness of methodology, quality of the analysis and the clarity of the final report. Data reliability and achievement of the study objectives have no significant impact on either the decision to accept or implement recommendations. The problem of symbolic use is discussed as a potential explanation for the observed results.

Confirmation of Paternity despite Three Genetic Incompatibilities at Chromosome 2.

DNA testing in cases of disputed paternity is a routine analysis carried out in genetic laboratories. The purpose of the test is to demonstrate similarities and differences in analyzed genetic markers between the alleged father, mother, and a child. The existence of differences in the examined loci between the child and the presumed father may indicate the exclusion of biological parenthood. However, another reason for such differences is genetic mutations, including chromosome aberrations and genome mutations. The presented results relate to genetic analyses carried out on three persons for the purposes of disputed paternity testing. A deviation from inheritance based on Mendel's Law was found in 7 out of 53 STR-type loci examined. All polymorphic loci that ruled out the paternity of the alleged father were located on chromosome 2. Additional analysis of 32 insertion-deletion markers (DIPplex, Qiagen) and sequencing of 94 polymorphic positions of the single nucleotide polymorphism (SNP) type (Illumina, ForenSeq) did not exclude the defendant's biological paternity. A sequence analysis of STR alleles and their flanking regions confirmed the hypothesis that the alleles on chromosome 2 of the child may originate only from the mother. The results of the tests did not allow exclusion of the paternity of the alleged father, but are an example of uniparental maternal disomy, which is briefly described in the literature.

Recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics on forensic Y chromosome typing.

Y chromosome typing has been performed in forensic genetic practice for more than 20 years. The latest recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) concerning the application of Y-chromosomal markers in forensic genetics were published in 2006. The aim of this report is to recapitulate, systematise and supplement existing recommendations on the forensic analysis of polymorphism of the Y chromosome with standards already implemented in practice, new capabilities linked to the development of research techniques as well as current solutions used in statistical analysis. The recommendations have been adapted specifically to aspects related to the preparation of expert opinions in the field of forensic genetics in Poland. The Polish Speaking Working Group of the ISFG believes that the presented guidelines should become a standard implemented by all Polish laboratories performing Y chromosome typing for forensic purposes.

Examination of LT-DNA traces - literature overview and general recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics (ISFG-PL).

The available literature on traces characterised by a suboptimal amount of DNA, as well as expert research practice, show the complex nature of LT-DNA traces: from their detection and collection, through genetic analysis, up to the interpretation of final results. The aims of this paper are to systematise the current state of knowledge on handling LT-DNA traces and develop examination guidelines, as recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics (ISFG-PL). The proposed guidelines should be followed by all Polish laboratories conducting forensic genetic analyses for the purpose of judicial proceedings.

Dual amplification strategy for improved efficiency of forensic DNA analysis using NGM Detect™, NGM™ or Globalfiler™ kits.

The new generation of STR amplification kits with improved sensitivity and additional genetic markers is designed particularly for analyzing difficult traces with a high DNA degradation index, presence of inhibitors and low level of DNA. In the new NGM Detect™ kit, modifications including changing the primers' sequences and shortening of STR markers are introduced. The quality control system (IQCS, IQCL) used to detect DNA degradation and the presence of inhibitors in the sample is an additional feature. The purpose of this study was to compare the results of analysis of different kinds of typical casework samples obtained using NGM™ or GlobalFiler™ kits with those generated using the new NGM Detect™ kit. The results indicate that the tested kit is particularly useful for the analysis of challenging samples for which incomplete profiles are generated with the NGM™ or GlobalFiler™ kits. The increased number of positively typed alleles gives better statistical parameters in genetic identification cases. We conclude that the NGM Detect™ kit can be recommended for the double amplification protocol together with the NGM or GlobalFiler™ kits.

Recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics for forensic mitochondrial DNA testing.

Although mitochondrial DNA (mtDNA) testing has been used in forensic genetics only since the mid-1990s, forensic DNA laboratories have been recently increasing the range of mtDNA sequencing, employing new analytical approaches and methods of data analysis. Therefore, it seems fitting to gather and systematize existing recommendations in the field of mtDNA analysis for forensic purposes, and formulate a set of interpretative guidelines which are especially relevant in view of recent developments in the forensic casework. The starting point is the recommendations of the International Society for Forensic Genetics (ISFG) which, in the opinion of the Polish Speaking Working Group of the ISFG (ISFG- PL), should be followed by all Polish laboratories conducting forensic testing.

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.

Genetic Identification of Communist Crimes' Victims (1944-1956) Based on the Analysis of One of Many Mass Graves Discovered on the Powazki Military Cemetery in Warsaw, Poland.

As the result of the communist terror in Poland, during years 1944-1956 more than 50,000 people died. Their bodies were buried secretly, and most places are still unknown. The research presents the results of identification of people buried in one of many mass graves, which were found at the cemetery Powazki Military in Warsaw, Poland. Exhumation revealed the remains of eight people, among which seven were identified genetically. Well-preserved molars were used for the study. Reference material was collected from the closest living relatives. In one case, an exhumation of victim's parents had to be performed. DNA from swabs was extracted with a PrepFiler® BTA Forensic DNA Extraction Kit and organic method. Autosomal, Y-STR amplification, and mtDNA sequencing were performed. The biostatistical calculations resulted in LR values from 1608 to 928 × 1018 . So far, remains of more than 50 victims were identified.

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.

Development of a forensically useful age prediction method based on DNA methylation analysis.

Forensic DNA phenotyping needs to be supplemented with age prediction to become a relevant source of information on human appearance. Recent progress in analysis of the human methylome has enabled selection of multiple candidate loci showing linear correlation with chronological age. Practical application in forensic science depends on successful validation of these potential age predictors. In this study, eight DNA methylation candidate loci were analysed using convenient and reliable pyrosequencing technology. A total number of 41 CpG sites was investigated in 420 samples collected from men and women aged from 2 to 75 years. The study confirmed correlation of all the investigated markers with human age. The five most significantly correlated CpG sites in ELOVL2 on 6p24.2, C1orf132 on 1q32.2, TRIM59 on 3q25.33, KLF14 on 7q32.3 and FHL2 on 2q12.2 were chosen to build a prediction model. This restriction allowed the technical analysis to be simplified without lowering the prediction accuracy significantly. Model parameters for a discovery set of 300 samples were R(2)=0.94 and the standard error of the estimate=4.5 years. An independent set of 120 samples was used to test the model performance. Mean absolute deviation for this testing set was 3.9 years. The number of correct predictions ±5 years achieved a very high level of 86.7% in the age category 2-19 and gradually decreased to 50% in the age category 60-75. The prediction model was deterministic for individuals belonging to these two extreme age categories. The developed method was implemented in a freely available online age prediction calculator.

Practical aspects of genetic identification of hallucinogenic and other poisonous mushrooms for clinical and forensic purposes.

AIM: To assess the usefulness of a DNA-based method for identifying mushroom species for application in forensic laboratory practice. METHODS: Two hundred twenty-one samples of clinical forensic material (dried mushrooms, food remains, stomach contents, feces, etc) were analyzed. ITS2 region of nuclear ribosomal DNA (nrDNA) was sequenced and the sequen-ces were compared with reference sequences collected from the National Center for Biotechnology Information gene bank (GenBank). Sporological identification of mushrooms was also performed for 57 samples of clinical material. RESULTS: Of 221 samples, positive sequencing results were obtained for 152 (69%). The highest percentage of positive results was obtained for samples of dried mushrooms (96%) and food remains (91%). Comparison with GenBank sequences enabled identification of all samples at least at the genus level. Most samples (90%) were identified at the level of species or a group of closely related species. Sporological and molecular identification were consistent at the level of species or genus for 30% of analyzed samples. CONCLUSION: Molecular analysis identified a larger number of species than sporological method. It proved to be suitable for analysis of evidential material (dried hallucinogenic mushrooms) in forensic genetic laboratories as well as to complement classical methods in the analysis of clinical material.

Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science.

Age estimation in forensic investigations may complement the prediction of externally visible characteristics and the inference of biogeographical ancestry, thus allowing a better description of an unknown individual. Multiple CpG sites that show linear correlation between age and degree of DNA methylation have been identified in the human genome, providing a selection of candidates for age prediction. In this study, we optimized an assay based on bisulfite conversion and pyrosequencing of 7 CpG sites located in the ELOVL2 gene. Examination of 303 blood samples collected from individuals aged 2-75 years allowed selection of the most informative site, explaining 83% of variation in age. The final linear regression model included two CpG sites in ELOVL2 and enabled age prediction with R(2)=0.859, prediction error=6.85 and mean absolute deviation MAD=5.03. Examination of a testing set of 124 blood samples (MAD=5.75) showed that 68.5% of samples were correctly predicted, assuming that chronological and predicted ages matched ± 7 years. It was found that the ELOVL2 methylation status in bloodstains had not changed significantly after 4 weeks of storage in room temperature conditions. Analysis of 45 bloodstains deposited on tissue paper after 5, 10 and 15 years of storage in room conditions indicated that although a gradual decrease of positive PCR results was observed, the general age prediction success rate remained similar and equaled 60-78%. The obtained results show that the ELOVL2 locus provides a very good source of information about human chronological age based on analysis of blood, including bloodstains, and it may constitute a powerful and reliable predictor in future forensic age estimation models.

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour.

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.

Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains.

BACKGROUND: DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person's externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. METHODS: Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. RESULTS: Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Wladyslaw Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. CONCLUSIONS: Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals.