Search

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler ® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R 2 , and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 μg/μL - 0.24 ng/μL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 μL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (Copyright © 2024 by PTMSiK.)

Background: D5S818 discrepancies have been reported in forensic parental testing due to null alleles. However, more cases may be ignored since proportional null alleles were missed without detection of heredity discrepancy between parents and offspring., Results: In this study, null allele 12 at D5S818 was detected by the PowerPlex ® 21 System with a higher occurrence rate on the basis of review on 2824 samples from the 1282 routine cases in Chinese Han population. Sequencing results revealed novel variant of guanine (G) into adenine (A) in the 7th [AGAT] repeats in the core repeat region accompanied by rs1187948322 in the samples with null allele 12., Conclusions: Forensic STR typing may benefit from this discovery: (1) primer design of CE profiling system could be improved for sensitive population and (2) polymorphic information could be enriched for the accuracy and precision of NGS genotyping system. Peak area of D5S818 was also analyzed through different commercial STR kits. It is suggested that more attention should be paid on observed homozygosity with reduced peak area, especially for the samples from Chinese Han population., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

In recent years, scientists have found a close correlation between DNA methylation and aging in epigenetics. With the in-depth research in the field of DNA methylation, researchers have established a quantitative statistical relationship to predict the individual ages. This work used human blood tissue samples to study the association between age and DNA methylation. We built two predictors based on healthy and disease data, respectively. For the health data, we retrieved a total of 1191 samples from four previous reports. By calculating the Pearson correlation coefficient between age and DNA methylation values, 111 age-related CpG sites were selected. Gradient boosting regression was utilized to build the predictive model and obtained the R 2 value of 0.86 and MAD of 3.90 years on testing dataset, which were better than other four regression methods as well as Horvath's results. For the disease data, 354 rheumatoid arthritis samples were retrieved from a previous study. Then, 45 CpG sites were selected to build the predictor and the corresponded MAD and R 2 were 3.11 years and 0.89 on the testing dataset respectively, which showed the robustness of our predictor. Our results were better than the ones from other four regression methods. Finally, we also analyzed the twenty-four common CpG sites in both healthy and disease datasets which illustrated the functional relevance of the selected CpG sites.

Description of a perpetrator's eye colour can be an important investigative lead in a forensic case with no apparent suspects. Herein, we present 11 SNPs (Eye Colour 11-EC11) that are important for eye colour prediction and eye colour prediction models for a two-category reporting system (blue and brown) and a three-category system (blue, intermediate, and brown). The EC11 SNPs were carefully selected from 44 pigmentary variants in seven genes previously found to be associated with eye colours in 757 Europeans (Danes, Swedes, and Italians). Mathematical models using three different reporting systems: a quantitative system (PIE-score), a two-category system (blue and brown), and a three-category system (blue, intermediate, brown) were used to rank the variants. SNPs with a sufficient mean variable importance (above 0.3%) were selected for EC11. Eye colour prediction models using the EC11 SNPs were developed using leave-one-out cross-validation (LOOCV) in an independent data set of 523 Norwegian individuals. Performance of the EC11 models for the two- and three-category system was compared with models based on the IrisPlex SNPs and the most important eye colour locus, rs12913832. We also compared model performances with the IrisPlex online tool (IrisPlex Web). The EC11 eye colour prediction models performed slightly better than the IrisPlex and rs12913832 models in all reporting systems and better than the IrisPlex Web in the three-category system. Three important points to consider prior to the implementation of eye colour prediction in a forensic genetic setting are discussed: (1) the reference population, (2) the SNP set, and (3) the reporting strategy.

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

One of the main goals of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. The GHEP-ISFG supports several Working Commissions which develop different scientific activities. One of them, the Working Commission on "Massively Parallel Sequencing (MPS): Forensic Applications", organized its first collaborative exercise on forensic applications of MPS technology in 2019. The aim of this exercise was to assess the concordance between the MPS results and those obtained with conventional technologies (capillary electrophoresis and Sanger sequencing), as well as to compare the results obtained within the different MPS platforms and/or the different kits/panels and analysis software packages (commercial and open-access) available on the market. The seven participating laboratories analyzed some samples of the annual GHEP-ISFG proficiency test (EIADN No. 27 (2019)), using Ion Torrent™ or MiSeq FGx® platforms. Six of them sent autosomal STR sequence data, five laboratories performed MPS analysis of individual identification SNPs, four laboratories reported MPS data of Y-chromosomal STRs, and X-chromosomal STRs, three laboratories performed MPS analysis of ancestry informative SNPs and phenotype informative SNPs, two labs performed MPS analysis of the mitochondrial DNA control region, and only one lab produced MPS data of lineage informative SNPs. Autosomal STR sequencing results were highly concordant to the consensus obtained by capillary electrophoresis in the EIADN No. 27 (2019) exercise. Furthermore, in general, a high level of concordance was observed between the results of the participating laboratories, regardless of the platform used. The main discordances were due to errors during the analysis process or from sequence data obtained with low depth of coverage. In this paper we highlight some issues that still arise, such as standardization of the nomenclature for STRs analyzed by sequencing with MPS, the universal uptake of a nomenclature framework by the analysis software, and well established validation and accreditation of the new MPS platforms for use in routine forensic case-work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Short tandem repeat (STR) markers play a significant role in genetic applications and have proved to be effective for the personal identification in forensic medicine. In this study, a six-dye multiplex composed of 23 autosomal STR loci (TH01, D3S1358, Penta D, D6S1043, D21S11, TPOX, D1S1656, D12S391, Penta E, D10S1248, D22S1045, D19S433, D8S1179, D2S1338, D2S441, D18S51, vWA, FGA, D16S539, CSF1PO, D13S317, D5S818, D7S820), one Y chromosome STR (DYS391), two internal quality control markers (Quality Sensor QS1 and QS2), and Amelogenin was evaluated., Methods: Evaluation studies, including PCR-based studies, sensitivity studies, species specificity studies, stability studies, DNA mixture studies, concordance studies, and precision evaluations were performed according to the guidelines of "Validation Guidelines for Forensic DNA Analysis Methods (2016)" by the Scientific Working Group on DNA Analysis Methods (SWGDAM). In addition, the forensic characteristics of 357 unrelated male samples from Han and Hui populations in China were investigated using 27 markers., Results: Full STR profiles were obtained from different reaction volumes (5 ~ 25 μl), cycle numbers (28 ~ 34 cycles) and annealing temperatures (58 ~ 62°C). All STR profiles were obtained at humic acid concentration of up to 200 ng/μl and hematin concentration of up to 500 μM. No peaks were observed in most common animal samples except two innovative internal PCR controls (Quality Sensor QS1 and QS2). The six-dye multiplex showed a notably high value for the combined probability of exclusion (CPE), exhibiting values of with 0.99999999977688 in the Han population and 0.999999999583875 in the Hui population. The values of combined probability of discrimination (CPD) were 0.999999999999999999999999999997453 in the Han population and 0. 999999999999999999999999999994398 in the Hui population. In addition, concordance studies showed that there was no difference with the AGCU Express Marker 22 Kit., Conclusion: The results indicated that the Investigator ® 26plex QS Kit is a robust, reliable, and suitable tool for forensic analysis and databasing., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Competing Interests: The authors have declared that no competing interests exist.

STRidER, the STRs for Identity ENFSI Reference Database, is a curated, freely publicly available online allele frequency database, quality control (QC) and software platform for autosomal Short Tandem Repeats (STRs) developed under the endorsement of the International Society for Forensic Genetics. Continuous updates comprise additional STR loci and populations in the frequency database and many further STR-related aspects. One significant innovation is the autosomal STR data QC provided prior to publication of datasets. Such scrutiny was lacking previously, leaving QC to authors, reviewers and editors, which led to an unacceptably high error rate in scientific papers. The results from scrutinizing 184 STR datasets containing >177,000 individual genotypes submitted in the first two years of STRidER QC since 2017 revealed that about two-thirds of the STR datasets were either being withdrawn by the authors after initial feedback or rejected based on a conservative error rate. Almost no error-free submissions were received, which clearly shows that centralized QC and data curation are essential to maintain the high-quality standard required in forensic genetics. While many errors had minor impact on the resulting allele frequencies, multiple error categories were commonly found within single datasets. Several datasets contained serious flaws. We discuss the factors that caused the errors to draw the attention to redundant pitfalls and thus contribute to better quality of autosomal STR datasets and allele frequency reports.

Circassians are a Caucasian ethnic group who make up a significant minority in Jordan. Although other ethnic groups have been the subject of forensic genetic analysis, no published study has investigated the forensic genetic efficiency of short tandem repeats (STRs) in Circassians, neither in Jordan nor in any other country. The main objective of the current study is to determine the allelic frequencies and evaluate the forensic efficiency parameters of 21 highly polymorphic autosomal STR loci among the Circassian subpopulation in Jordan. The GlobalFiler loci were amplified using DNA extracted from the whole blood samples of 150 Jordanian Circassians. The SE33 locus was found to be the most informative and polymorphic STR marker while TPOX was the least informative. However, allele 8 of TPOX was the most common across all of the investigated 21 loci in Jordanian Circassians. The combined matching probability (CMP) and combined power of discrimination (CPD) were 5.02E-24 and 0.9999999, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Genetic polymorphisms at 23 short tandem repeat (STR) loci were investigated in 1,215 Jining Han individuals from Jining city, Shandong province, eastern China., Methods: We used population genetic data of 23 autosomal STR loci included in the Huaxia Platinum system to evaluate 1,215 unrelated Chinese Han individuals in the Jining Han population. Allele frequencies and forensic parameters of the STR loci were determined and genetic relationships among the Jining Han and other Chinese populations were evaluated., Results: In total, we observed 321 alleles, with frequencies ranging from 0.00041 to 0.52222. The combined discrimination power and probability of excluding paternity were 0.99999999999999999999999999919 and 0.99999999962, respectively. No deviations from HWE were observed at any loci. Population comparisons showed that the Xinjiang groups (Uyghur and Kazakh) and the Mongolian and Tibetan groups were isolated, while the Jining Han population clustered together with other populations, except the Guizhou Han population., Conclusion: This study demonstrated that 23 autosomal STR loci included in the Huaxia Platinum system are highly polymorphic and suitable for personal forensic identification and paternity testing in this population., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: Short tandem repeats (STRs) are essential genetic markers for forensic applications and population estimations; thus the population genetics of STR loci have been extensively studied and discussed., Methods: In the present study, we detected 21 autosomal noncombined DNA index system (non-CODIS) STR loci in a Chinese Han population from Shanghai, calculated their forensic parameters and analyzed their genetic relationships with reported reference populations in mainland China., Results: A total of 173 alleles were observed, with corresponding allele frequencies from 0.0020 to 0.5512. The cumulative power of discrimination (CPD) and the cumulative probability of exclusion (CPE) values of the 21 STR loci were 0.999999999999999999997337058271 and 0.99999953732495, respectively. The results of interpopulation differentiation, phylogenetic, multidimensional scaling, and structure analyses indicated a closer genetic relationship of the studied population with Han populations from other regions of China than with other populations., Conclusions: The 21 STR loci exhibited high genetic polymorphism in the studied Shanghai_Han population and could be used for forensic applications and population genetic studies., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: Individual identification is one of the most important tasks in the field of forensic genetics. Insertion/Deletion (InDel) polymorphism marker has been a promising marker for individual identification. However, a part of InDel loci in commonly used commercial kit show low polymorphisms in Chinese populations., Methods: We evaluated a panel of 35 InDel loci constructed previously for individual identifications in Hui group. Subsequently, population data of three Chinese populations from 1,000 Genomes Project database were used to evaluate individual identification performance of these 35 InDels. Forensic parameters, such as heterozygosity, power of exclusion, match probability and power of discrimination, were calculated to evaluate the forensic efficiency of these loci in Hui group. The heatmap of insertion allelic frequencies, Nei's genetic distances, pairwise fixation index values, principal component analyses and admixture analyses were used to analyze the genetic differentiations and structure between Hui group and other populations., Results: In studied Hui group, besides rs3054057, polymorphism information content values of the remaining loci were greater than 0.3. Values of expected heterozygosity of these loci were close to 0.5. The combined power of discrimination and power of exclusion values were 0.99999999999999659609 and 0.998682, respectively. Analyses of population genetics revealed that Chinese Hui group had closer genetic relationships with East Asian populations than other intercontinental populations., Conclusion: The forensic statistical analyses revealed these loci showed relatively high genetic polymorphisms in Chinese Hui group, and could be served as a useful tool for individual identifications in Hui group. Population genetic evaluations indicated that Chinese Hui group had close genetic relationships with East Asian populations., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

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.

Sudden cardiac death (SCD) is often associated with structural abnormalities of the heart during autopsy. This study sought to compare the diagnostic yield of postmortem genetic testing in (1) cases with structural findings of uncertain significance at autopsy to (2) cases with autopsy findings diagnostic of cardiomyopathy. We evaluated 57 SCD cases with structural findings at cardiac autopsy. Next-generation sequencing using a panel of 77 primary electrical disorder and cardiomyopathy genes was performed. Pathogenic and likely pathogenic variants were classified using American College of Medical Genetics (ACMG) consensus guidelines. In 29 cases (51%) autopsy findings of uncertain significance were identified whereas in 28 cases (49%) a diagnosis of cardiomyopathy was established. We identified a pathogenic or likely pathogenic variant in 10 cases (18%); in 1 (3%) case with non-specific autopsy findings compared with 9 (32%) cases with autopsy findings diagnostic of cardiomyopathy (p = 0.0054). The yield of genetic testing in SCD cases with autopsy findings consistent with cardiomyopathy is comparable with the yield in cardiomyopathy patients that are alive. Genetic testing in cases with findings of uncertain significance offers lower clinical utility than in cardiomyopathy, with lower yields than detected previously. This highlights the need for stringent evaluation of variant pathogenicity.

Y-chromosomal short tandem repeats (Y-STRs) have proven to be very useful in investigating sexual assault cases and in paternity lineage differentiation. However, currently available commercial Y-STR multiplex amplification systems bear the limitations in the identification of related males from the same paternal lineage due to there being an insufficient number of loci in any single amplification kit. The aim of this study was to establish and validate a novel 6-dye, 36-plex Y-STR multiplex amplification system that incorporated all of the loci present in the Yfiler™ Plus kit (DYS19, DYS385a/b, DYF387S1, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, Y_GATA_H4) as well as a further nine highly polymorphic Y-STR loci (DYS388, DYS444, DYS447, DYS522, DYS527a/b, DYS549, DYS596, DYS643). The novel system was optimized and validated by a series of studies that tested the effect of different PCR-based conditions as well as the species specificity, sensitivity, stability, stutter precision, suitability for use on DNA mixtures, reproducibility, and parallel testing of the system, as well as its performance on casework samples and population analysis, according to the SWGDAM developmental validation guidelines. A total of 246 haplotypes were found for the 36 Y-STRs among 247 Guangdong Han unrelated males. Collectively, the results demonstrate that the developed 36-plex Y-STR system is sensitive, robust, reliable, and highly informative for use in forensic genetics.

Background: The analysis of Y-SNPs from crime scene samples is helpful for investigators in narrowing down suspects by predicting biogeographical ancestry., Objective: In this study, a PCR-reverse blot hybridization assay (REBA) for predicting Y-chromosome haplogroups was employed to determine the major haplogroups worldwide, including AB, DE, C, C3, F, K, NO, O, O2, and O3 and evaluated., Methods: The REBA detects nine biallelic Y chromosome markers (M9, M89, M122, M145, M175, M214, M217, P31, and RPS4Y 711 ) simultaneously using multiple probes., Results: The REBA for Y-single nucleotide polymorphisms (SNP) genotyping was performed using 40 DNA samples from Asians-14 Koreans, 10 Indonesians, six Chineses, six Thais, and four Mongolians. 40 Asian samples were identified as haplogroup O2 (40%), O3 (32.5%), C3 (17.5%), O (7.5%) and K (2.5%). These cases were confirmed by DNA sequence analysis (κ = 1.00; P < 0.001)., Conclusion: PCR-REBA is a rapid and reliable method that complements other SNP detection methods. Therefore, implementing REBA for Y-SNP testing may be a useful tool in predicting Y-chromosome haplogroups.

Sharing data between forensic scientists on DNA transfer, persistence, prevalence and recovery (TPPR) is crucial to advance the understanding of these issues in the criminal justice community. We present the results of a collaborative exercise on reporting forensic genetics findings given activity level propositions. This exercise outlined differences in the methodology that was applied by the participating laboratories, as well as limitations to the use of published data on DNA TPPR. We demonstrate how publication of experimental results in scientific journals can be further improved to allow for an adequate use of these data. Steps that can be taken to share and use these data for research and casework purposes are outlined, and the prospects for future sharing of data through publicly accessible databases are discussed. This paper also explores potential avenues to proceed with implementation and is intended to fuel the discussion on sharing data pertaining to DNA TPPR issues. It is further suggested that international standardization and harmonization on these topics will benefit the forensic DNA community as it has been achieved in the past with the harmonization of STR typing systems., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

MIX13 was an interlaboratory exercise directed by NIST in 2013. The goal of the exercise was to evaluate the general state of interpretation methods in use at the time across the forensic community within the US and Canada and to measure the consistency in mixture interpretation. The findings were that there was a large variation in analysts' interpretations between and within laboratories. Within this work, we sought to evaluate the same mock mixture cases analyzed in MIX13 but with a more current view of the state-of-the-science. Each of the five cases were analyzed using the Identifiler™ multiplex and interpreted with the combined probability of inclusion, CPI, and four different modern probabilistic genotyping systems. Cases 1-4 can be interpreted without difficulty by any of the four PG systems examined. Cases 1 and 4 could also be interpreted successfully with the CPI by assuming two donors. Cases 2 and 3 cannot be interpreted successfully with the CPI because of potential of allele dropout. Case 3 demonstrated the need to consider relevant background information before interpretation of the profile. This case does not show that there is some barrier to interpretation caused by relatedness beyond the increased allelic overlap that can occur. Had this profile been of better template it might have been interpreted using the CPI despite the (potential) relatedness of contributors. Case 5 suffers from over-engineering. It is unclear whether reference 5C, a non-donor, can be excluded by manual methods. Inclusion of reference 5C should be termed an adventitious match not a false inclusion. Beyond this statement this case does not contribute to the interlaboratory study of analyst/laboratory interpretation method performance, instead, it explores the limits of DNA analysis. Taken collectively the analysis of these five cases demonstrates the benefits of changing from CPI to a PG system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Interlaboratory studies are a type of collaborative exercise in which many laboratories are presented with the same set of data to interpret, and the results they produce are examined to get a "big picture" view of the effectiveness and accuracy of analytical protocols used across participating laboratories. In 2005 and again in 2013, the Applied Genetics Group of the National Institute of Standards and Technology (NIST) conducted interlaboratory studies involving DNA mixture interpretation. In the 2005 NIST MIX05 study, 69 laboratories interpreted data in the form of electropherograms of two-person DNA mixtures representing four different mock sexual assault cases with different contributor ratios. In the 2013 NIST MIX13 study,108 laboratories interpreted electropherogram data for five different case scenarios involving two, three, or four contributors, with some of the contributors potentially related. This paper describes the design of these studies, the variations observed among laboratory results, and lessons learned., (Published by Elsevier B.V.)

The interpretation of evidence continues to be one of the biggest challenges facing the forensic community. This is the first of two papers intended to provide advice on difficult aspects of evaluation and in particular on the formulation of propositions. The scientist has a dual role: investigator (crime-focused), where often there is no suspect available and a database search may be required; evaluator (suspect-focused), where the strength of evidence is assessed in the context of the case. In investigative mode, generally the aim is to produce leads regarding the source of the DNA. Sub-source level propositions will be adequate to help identify potential suspects who can be further investigated by the authorities. Once in evaluative mode, given the defence version of events of the person of interest, it may become necessary to consider alternatives that go beyond the source of the DNA (i.e., to consider activity level propositions). In the evaluation phase, it is crucial that formulation of propositions allows the assessment of all the results that will help with the issue at hand. Propositions should therefore be precise (indication of the number of contributors, information on the relevant population etc.), be about causes, not effects (e.g. a 'matching' DNA profile) and to avoid bias, must not be findings-led. This means that ideally, propositions should be decided based on the case information and before the results of the comparisons are known. This paper primarily reflects upon what has been coined as "sub-source level propositions". These are restricted to the evaluation of the DNA profiles themselves, and help answer the issue regarding the source of the DNA. It is to be emphasised that likelihood ratios given sub-source level propositions cannot be carried over to a different level - for example, activity level propositions, where the DNA evidence is put into the context of the alleged activities. This would be highly misleading and could give rise to miscarriages of justice; this will be discussed in a second paper. The value of forensic results depends not only on propositions, but also on the type of results (e.g. allelic designations, peak heights, replicates) and upon the model used: it is therefore important to discuss these aspects. Finally, since communication is key to help understanding by courts, we will explore how to convey the value of the results and explain the importance of avoiding the practice of transposing the conditional., (Copyright © 2018 Elsevier B.V. All rights reserved.)

One of the main goals of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, six exercises have been organized. At the present edition (GHEP-MIX06), with 25 participant laboratories, the exercise main aim was to assess mixture profiles results by issuing a report, from the proposal of a complex mock case. One of the conclusions obtained from this exercise is the increasing tendency of participating laboratories to validate DNA mixture profiles analysis following international recommendations. However, the results have shown some differences among them regarding the edition and also the interpretation of mixture profiles. Besides, although the last revision of ISO/IEC 17025:2017 gives indications of how results should be reported, not all laboratories strictly follow their recommendations. Regarding the statistical aspect, all those laboratories that have performed statistical evaluation of the data have employed the likelihood ratio (LR) as a parameter to evaluate the statistical compatibility. However, LR values obtained show a wide range of variation. This fact could not be attributed to the software employed, since the vast majority of laboratories that performed LR calculation employed the same software (LRmixStudio). Thus, the final allelic composition of the edited mixture profile and the parameters employed in the software could explain this data dispersion. This highlights the need, for each laboratory, to define through internal validations its criteria for editing and interpreting mixtures, and to continuous train in software handling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (µPCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 µl each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the µPCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both µPCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis.

The STR sequence template file published in 2016 as part of the considerations from the DNA Commission of the International Society for Forensic Genetics on minimal STR sequence nomenclature requirements, has been comprehensively revised and audited using the latest GRCh38 genome assembly. The list of forensic STRs characterized was expanded by including supplementary autosomal, X- and Y-chromosome microsatellites in less common use for routine DNA profiling, but some likely to be adopted in future massively parallel sequencing (MPS) STR panels. We outline several aspects of sequence alignment and annotation that required care and attention to detail when comparing sequences to GRCh37 and GRCh38 assemblies, as well as the necessary matching of MPS-based allele descriptions to previously established repeat region structures described in initial sequencing studies of the less well known forensic STRs. The revised sequence guide is now available in a dynamically updated FTP format from the STRidER website with a date-stamped change log to allow users to explore their own MPS data with the most up-to-date forensic STR sequence information compiled in a simple guide., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Environmental forensics is a tool that uses chemical, physical, and statistical techniques to investigate contaminants in the environment as a means to determine attribution for legal purposes. Environmental microbiology is a branch of science that has benefited from the use of metagenomics. The term microbial forensics, which includes nucleic acid sequencing methods, is now used to investigate the sources of microorganisms for attribution purposes as well. Environmental microbial forensics can fully address the questions that must be answered for attribution of causation and subsequent remedial actions within a reasonably short time frame. Although sensu stricto forensics refers to obtaining scientific evidence to be presented during legal proceedings, the term sensu lato is used as a description of the procedures used to reconstruct previous events, such as contamination. The term microbial forensics was first used to describe a forensic science approach for attribution purposes, specifically for bioterror as a purposeful release of pathogen microorganisms, but it also especially refers to investigations on the inadvertent or accidental release of pathogenic agents. However, microbial forensics can be used to determine the source of a microorganism or a group of microorganisms, regardless of whether they are pathogenic or not. Microbial forensics has limitations, but it should be used as part of a toolbox of methods to be relied upon when doing forensic studies. Environmental microbial forensics can only benefit from the development of new methods, and we already are experiencing a paradigm change in terms of approaches to the forensic sciences.

Microbiome analysis of environmental samples may represent the next frontier in environmental microbial forensics. Next-generation sequencing technologies significantly increased the available genetic data that could be used as evidentiary material. It is not clear, however, whether the microbiome can scale across institutions using forensic-based evidence due to the data resource requirements and the associated costs of maintaining these databases. A successful microbiome study is impacted by the quality of the information gathered and the steps in sample processing and data analysis. To ascertain the validity of methods and the results obtained, there needs to be a stringent procedure to validate the methods and ensure that the results are comparable and reproducible, not only within the laboratory but also between laboratories conducting similar research. Of primary importance for meaningful microbiome studies is an experimental design that leads to carefully executed, controlled, and reproducible studies. The microbiome literature contains a fair share of anecdotal descriptions of microbial community composition and "diagnostic" relative abundance of the taxa therein. These studies are now being supplemented by experimental designs that feature repeated measurements, error estimates, correlations of microbiota with covariates, and increasingly sophisticated statistical tests that enhance the robustness of data analysis and study conclusions. It is imperative to be careful, especially when carrying out attribution studies, to be fully aware of the possible biases included in a specific sample being analyzed.

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

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.

The beginnings of forensic genetics, one of the most rapidly growing fields of research, can be traced to Great Britain in 1985. It appeared in Poland in 1989. It uses the most advanced technologies, including the investigation of the variability of the human genome through mass parallel sequencing, which help, among other things, to analyze features of human appearance and origin. These technologies coexist with well standardized techniques of multiplex short tandem repeat analysis based on capillary electrophoresis, which allows to obtain a unique individual profile at a minimal cost. Legislation, research standards and guidelines developed by opinion-forming institutions and expert teams play a key role in the field of genetic forensic examinations. This study presents the current normative state of this area. It precedes the presentation of guidelines concerning the main aspects of research in the field of forensic genetics in Poland, prepared by a team of experts gathered within the Polish Speaking Working Group of the International Forensic Genetics Society and the Forensic Genetics Committee of the Polish Society of Forensic Medicine and Criminology.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina's MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina's FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.

The STR Sequencing Project (STRSeq) was initiated to facilitate the description of sequence-based alleles at the Short Tandem Repeat (STR) loci targeted in human identification assays. This international collaborative effort, which has been endorsed by the ISFG DNA Commission, provides a framework for communication among laboratories. The initial data used to populate the project are the aggregate alleles observed in targeted sequencing studies across four laboratories: National Institute of Standards and Technology (N=1786), Kings College London (N=1043), University of North Texas Health Sciences Center (N=839), and University of Santiago de Compostela (N=944), for a total of 4612 individuals. STRSeq data are maintained as GenBank records at the U.S. National Center for Biotechnology Information (NCBI), which participates in a daily data exchange with the DNA DataBank of Japan (DDBJ) and the European Nucleotide Archive (ENA). Each GenBank record contains the observed sequence of a STR region, annotation ("bracketing") of the repeat region and flanking region polymorphisms, information regarding the sequencing assay and data quality, and backward compatible length-based allele designation. STRSeq GenBank records are organized within a BioProject at NCBI (https://www.ncbi.nlm.nih.gov/bioproject/380127), which is sub-divided into: commonly used autosomal STRs, alternate autosomal STRs, Y-chromosomal STRs, and X-chromosomal STRs. Each of these categories is further divided into locus-specific BioProjects. The BioProject hierarchy facilitates access to the GenBank records by browsing, BLAST searching, or ftp download. Future plans include user interface tools at strseq.nist.gov, a pathway for submission of additional allele records by laboratories performing population sample sequencing and interaction with the STRidER web portal for quality control (http://strider.online)., (Published by Elsevier B.V.)

DNA analysis was first applied to the identification of victims of armed conflicts and other situations of violence (ACOSV) in the mid-1990s, starting in South America and the Balkans. Argentina was the first country to establish a genetic database specifically developed to identify disappeared children. Following on from these programs the early 2000s marked major programs, using a largely DNA-led approach, identifying missing persons in the Balkans and following the attack on the World Trade Center in New York. These two identification programs significantly expanded the magnitude of events to which DNA analysis was used to help provide the identity of missing persons. Guidelines developed by Interpol (2014) [1] related to best practice for identification of human remains following DVI type scenarios have been widely disseminated around the forensic community; in numerous cases these guidelines have been adopted or incorporated into national guidelines/standards/practice. However, given the complexity of many humanitarian contexts in which forensic science is employed there is a lack of internationally accepted guidelines, related to these contexts, for authorities to reference. In response the Argentine government's Human Rights Division in the Ministry of Foreign Affairs and Worship (MREC) proposed that the United Nations (UN) should promote best practice in the use of forensic genetics in humanitarian forensic action: this was adopted by the UN in Resolutions A/HRC/RES/10/26 and A/HRC/RES/15/5. Following on from the adoption of the resolutions MREC has coordinated, with the support of the International Committee of the Red Cross (ICRC), the drafting of a set of guidelines (MREC, ICRC, 2014) [2], with input from national and international agencies. To date the guidelines have been presented to South America's MERCOSUR and the UN and have been disseminated to interested parties., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Forensic genetic laboratories perform an increasing amount of genetic analyses of the X chromosome, in particular to solve complex cases of kinship analysis. For some biological relationships X-chromosomal markers can be more informative than autosomal markers, and there are a large number of markers, methods and databases that have been described for forensic use. Due to their particular mode of inheritance, and their physical location on a single chromosome, some specific considerations are required when estimating the weight of evidence for X-chromosomal marker DNA data. The DNA Commission of the International Society for Forensic Genetics (ISFG) hereby presents guidelines and recommendations for the use of X-chromosomal markers in kinship analysis with a special focus on the biostatistical evaluation. Linkage and linkage disequilibrium (association of alleles) are of special importance for such evaluations and these concepts and the implications for likelihood calculations are described in more detail. Furthermore it is important to use appropriate computer software that accounts for linkage and linkage disequilibrium among loci, as well as for mutations. Even though some software exist, there is still a need for further improvement of dedicated software., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeq TM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9-10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as "low coverage" (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeq TM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1-44.8% and 10.9-58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Expressmarker mtDNA-SNP60 kit, a 3-dye fluorescence labeling forensic mitochondrial-SNP kit, was developed and modified based on previous work. In this study, it was validated through sensitivity, precision, accuracy, reproducibility, species specificity, mixture samples, PCR-based conditions, and inhibitor studies, according to the Scientific Working Group on DNA Analysis Methods guidelines. In the sensitivity study, full profile was obtained from template DNA with the quantity ≥ 4 pg. Consistent profiles were obtained at three different laboratories, which demonstrated the reproducibility of the kit. No peak was observed in the profiles of common animal species and microorganisms demonstrating high species specificity of the kit. In the mixture study, some loci (minor contributors) were missing at 19:1 and 9:1 ratios, whereas all loci could be profiled at other ratios (4:1, 2:1, and 1:1). PCR-based studies demonstrated that there recommended PCR conditions were optimized for the kit, and there was acceptable tolerance of error. The inhibitor study indicated that the kit has varying degrees of resistance to the presence of common inhibitors. Twelve random individuals were profiled by next-generation sequencing to confirm the allele-specific PCR assay, and a consistent result was obtained. Furthermore, 520 individuals from 260 families were profiled and the results showed that all loci of the confirmed mother and child were matched. In addition, forensic population genetics parameters, including random match probability, discrimination power, and haplotype diversity, were calculated (0.0474, 0.9526, and 0.9563, respectively). Our study demonstrates that the Expressmarker mtDNA-SNP60 kit can be a useful supplementary tool for forensic application., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In recent years statistical models for the analysis of complex (low-template and/or mixed) DNA profiles have moved from using only presence/absence information about allelic peaks in an electropherogram, to quantitative use of peak heights. This is challenging because peak heights are very variable and affected by a number of factors. We present a new peak-height model with important novel features, including over- and double-stutter, and a new approach to dropin. Our model is incorporated in open-source R code likeLTD. We apply it to 108 laboratory-generated crime-scene profiles and demonstrate techniques of model validation that are novel in the field. We use the results to explore the benefits of modeling peak heights, finding that it is not always advantageous, and to assess the merits of pre-extraction replication. We also introduce an approximation that can reduce computational complexity when there are multiple low-level contributors who are not of interest to the investigation, and we present a simple approximate adjustment for linkage between loci, making it possible to accommodate linkage when evaluating complex DNA profiles.

The HID-Ion AmpliSeq™ Identity Panel is a next-generation sequencing assay with 90 autosomal and 34 Y-chromosome SNPs that are amplified in one PCR step and subsequently sequenced using the Ion Personal Genome Machine (Ion PGM™) System. This assay was validated for relationship testing in our ISO 17025 accredited laboratory in 2015. Here, the essential parts of the validation report submitted to the Danish Accreditation Fund are presented. A total of 100 unrelated Danes were typed in duplicates and the locus balance, heterozygote balance (Hb) and noise levels were analysed in detail. Two loci were disregarded for casework because genotyping was uncertain. Hb for rs7520386 was skewed and high levels of noise were observed in rs576261. Three general acceptance criteria for analysis of single-source samples were defined: (i) sequencing depth > 200 reads, (ii) noise level < 3% and (iii) Hb > 0.3. A Python script named SNPonPGM was developed to assist the analyst by highlighting loci that do not fulfil the general acceptance criteria. Furthermore, SNPonPGM has functions that reduce the hands-on time of the reporting officer to a few minutes per case. Mixtures with DNA from two individuals in a 1:24 ratio were readily identified using the three criteria and the SNPonPGM script., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the progress and development of the DNA test and imaging technique, and the evolution of evidence rule which bring the discussions about whether the individual identification using imaging data is outdated, and other disputes such as whether radiologic evidence could be suitable for contemporary evidence and be used to solve the posture difference of imaging test. This article summaries the domestic and foreign researches of individual identification using imaging data in the past 20 years and reviews the problems above., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Currently, two of the most widely used X-chromosome STR (X-STR) multiplexes are composed by ten (GHEP-ISFG decaplex) and 12 markers (Investigator Argus X-12 Kit). The number of markers included is a drawback for complex relative testing cases, likewise the large size of some amplicons difficult their application to degraded samples. Here, we present a new multiplex of 17 X-STRs with the aim of increasing both the resolution power and forensic applicability. This newly proposed set includes the X-STRs of the GHEP-ISFG decaplex, four X-STRs from the Investigator Argus X-12 Kit, three of them also included in the decaplex, and six additional more. In order to ensure the allele designation, an allelic ladder was developed. The validation of the present multiplex was carried out according to the revised guidelines by the SWGDAM (Scientific Working Group on DNA Analysis Methods). A total of 488 unrelated individuals from four different continents were analyzed. The forensic efficiency evaluation showed high values of combined power of discrimination in males (≥0.999999996) and females (≥0.999999999999995) as well as combined paternity exclusion probabilities in trios (≥0.99999998) and duos (≥0.999996). The results presented herein have demonstrated that the new 17 X-STR set constitutes a high-resolution alternative to the current X-STR multiplexes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hen harrier (Circus cyaneus) is a bird of prey which is heavily persecuted in the UK because it preys on the game bird red grouse (Lagopus lagopus scoticus). To help investigations into illegal killings of hen harrier, a STR multiplex kit containing eight short tandem repeat (STR) markers and a chromohelicase DNA binding protein 1 (CHD 1) sexing marker was developed. The multiplex kit was tested for species specificity, sensitivity, robustness, precision, accuracy and stability. Full profiles were obtained with as little as 0.25 ng of template DNA. Concurrent development of an allelic ladder to ensure reliable and accurate allele designation across laboratories makes the SkydancerPlex the first forensic DNA profiling system in a species of wildlife to be fully validated according to SWGDAM and ISFG recommendations. An average profile frequency of 3.67 × 10(-8), a PID estimate of 5.3 × 10(-9) and a PID-SIB estimate of 9.7 × 10(-4) make the SkydancerPlex an extremely powerful kit for individualisation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

A 31-plex SNaPshot assay, named 'Global AIMs Nano', has been developed by reassembling the most differentiated markers of the EUROFORGEN Global AIM-SNP set. The SNPs include three tri-allelic loci and were selected with the goal of maintaining a balanced differentiation of: Africans, Europeans, East Asians, Oceanians and Native Americans. The Global AIMs Nano SNP set provides higher divergence between each of the five continental population groups than previous small-scale AIM sets developed for forensic ancestry analysis with SNaPshot. Both of these characteristics minimise potential bias when estimating co-ancestry proportions in individuals with admixed ancestry; more likely to be observed when using markers disproportionately informative for only certain population group comparisons. The optimised multiplex is designed to be easily implemented using standard capillary electrophoresis regimes and has been used to successfully genotype challenging forensic samples from highly degraded material with low level DNA. The ancestry predictive performance of the Global AIMs Nano set has been evaluated by the analysis of samples previously characterised with larger AIM sets., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)