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MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression. miRNAs have tissue-specific expression and are also present in various extracellular body fluids, including blood, tears, semen, vaginal fluid, and urine. Additionally, the expression of miRNAs in body fluids is linked to various pathological diseases, including cancer and neurodegenerative diseases. Examination of body fluids is important in forensic medicine as they serve as a valuable form of evidence. Due to its stability, miRNA offers an advantage for body fluid identification, which can be detected even after several months or from compromised samples. Identification of unique miRNA profiles for different body fluids enables the identification of the body fluids. Furthermore, miRNAs profiling can be used to estimate post-mortem interval. Various biochemical and molecular methods used for the identification of miRNAs have shown promising results. We discuss different miRNAs as specific biomarkers and their clinical importance in different pathological conditions, as well as their medicolegal importance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples., (Copyright © 2021 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

In terms of crime scene investigations by means of forensic DNA-analyses, burglaries are the number one mass crime in Switzerland. Around one third of the DNA trace profiles registered in the Swiss DNA database are related to burglaries. However, during the collection of potential DNA traces within someone's residence after a burglary, it is not known whether the sampled DNA originated from the perpetrator or from an inhabitant of said home. Because of the high incidence of burglaries, crime scene investigators usually do not collect reference samples from all the residents for economical and administrative reasons. Therefore, the presumably high probability that a DNA profile belonging to a person authorized to be at the crime scene ends up being sent to a DNA database for comparison, has to be taken into account. To our knowledge, no investigation has been made to evaluate the percentage of these non-perpetrator profiles straying into DNA databases. To shed light on this question, we collected reference samples from residents who had been victims of recent burglaries in their private homes. By comparing the profiles established from these reference samples with the profiles generated from trace DNA, we can show that the majority of the DNA samples collected in burglary investigations belong to the residents. Despite the limited number of cases included in the study, presumably due to a crime decline caused by the pandemic, we further show that trace DNA collection in the vicinity of the break and entry area, in particular window and door glasses, is most promising for sampling perpetrator instead of inhabitant DNA.

When DNA from a suspect is detected in a sample collected at a crime scene, there can be alternative explanations about the activity that may have led to the transfer, persistence and recovery of his/her DNA. Previous studies have shown that DNA can be indirectly transferred via intermediate surfaces and that DNA on a previously used object can persist after subsequent use of another individual. In addition, it has been shown that a person's shedder status may influence transfer, persistence, prevalence, and recovery of DNA. In this study we have investigated transfer persistence and recovery on zip-lock bags and tape, which are commonly encountered in drug cases and how the shedder status of the participants influenced the results. A probabilistic framework was developed which was based on a previously described Bayesian network with case-specific modifications. Continuous modelling of data was used to inform the Bayesian networks and two case scenarios were investigated. In the specific scenarios only moderate to low support for H p was obtained. Applying a continuous model based on the profile quality can change the LRs.

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3931 autosomal SNPs for extended kinship analysis, and no clinically relevant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict first to fifth degree relationships with strong statistical support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5000 SNP panel for forensic applications.

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories., (© 2021. The Author(s).)

Greater scrutiny and demands for innovation and increased productivity place pressures on scientists. Forensic genetics is advancing at a rapid pace but can only do so responsibly, usefully, and acceptably within ethical and legal boundaries. We argue that such boundaries require that forensic scientists embrace 'ethics as lived practice'. As a starting point, we critically discuss 'thin' ethics in forensic genetics, which lead to a myopic focus on procedures, and to seeing 'privacy' as the sole ethical concern and technology as a mere tool. To overcome 'thin' ethics in forensic genetics, we instead propose understanding ethics as an intrinsic part of the lived practice of a scientist. Therefore, we explore, within the context of three case studies of emerging forensic genetics technologies, ethical aspects of decision-making in forensic genetics research and in technology use. We discuss the creation, curation, and use of databases, and the need to engage with societal and policing contexts of forensic practice. We argue that open communication is a vital ethical aspect. Adoption of 'ethics as lived practice' supports the development of anticipatory capacity-empowering scientists to understand, and act within ethical and legal boundaries, incorporating the operational and societal impacts of their daily decisions, and making visible ethical decision making in scientific practice.

Dogs (Canis lupus familiaris) are among the most common companion animals in the Republic of Korea. Recently, there have been many criminal cases of dog cruelty, injury, and theft, among others. This has increased the importance of dog-related biological evidence at crime scenes. The National Forensic Service of the Republic of Korea conducts short tandem repeat (STR) analysis using the Thermo Scientific Canine Genotypes™ Panel 2.1 Kit (Canine Kit) to identify individual dogs through forensic analysis. The Canine Kit was developed as a forensic STR kit for the identification of individual dogs. However, an allelic ladder was neither developed nor included in the commercial kit, leaving an issue of accurate genotyping. Primer details for the 18 markers used in the Canine Kit are proprietary information, and thus, unavailable to end-users. In this study, an allelic ladder was constructed with 160 fragments by combining 158 fragments of STR alleles obtained by nested PCR and two fragments artificially obtained from the sex-determination marker. By including the new allelic ladder in analysis of samples amplified with the Canine Kit, the accuracy and reliability of data analysis were improved. Application of this allelic ladder would be helpful for interlaboratory data sharing and standardization of canine genotype databases., (© 2021 American Academy of Forensic Sciences.)

Background: Allele frequency using short tandem repeats (STRs) is used to calculate likelihood ratio for database match, to interpret DNA mixture and to estimate ethnic groups in forensic genetics. In Korea, three population studies for 23 STR loci have been conducted with different sample size for forensic purposes., Objective: We performed comparative analysis to determine how the difference of sample size affects the allele frequency and allele variation within same ethnic population (i.e. Korean). Furthermore, this study was conducted to check how the sampling group and multiplex kit also affect allele variation such as rare alleles and population specific alleles., Methods: To compare allele variation, we used allele frequencies of three population data published from three Korean forensic research groups. Allele frequencies were calculated using different sample sizes and multiplex kits: 526, 1000, and 2000 individuals, respectively., Results: The results showed the different distribution of allele frequencies in some loci. There was also a difference in the number of rare alleles observed by the sample size and sampling bias. In particular, an allele of 9.1 in the D2S441 locus was not observed in population study with 526 individuals due to multiplex kits., Conclusion: Because the allele frequencies play an important role in forensic genetics, even if the samples are derived from the same population, it is important to consider the effects of sample size, sampling bias, and selection of multiplex kits in population studies., (© 2021. The Author(s).)

Identifying the species involved in shark bite incidents is an ongoing challenge but is important to mitigate risk. We developed a sampling protocol to identify shark species from DNA transferred to inanimate objects during bite incidents. To develop and refine the technique, we swabbed shark bite impressions on surfboards and wetsuit neoprene collected under semicontrolled conditions. Methods were tested experimentally and then successfully used to identify the species involved in a real-world shark bite incident. Thirty-two of 33 bite impressions yielded sufficient DNA sequences for species identification, producing barcodes from five test species, including dusky, Galapagos, bull, tiger, and white shark. The latter three species collectively account for a majority of shark bites worldwide. Our method successfully identified the species (Galeocerdo cuvier) responsible for a fatal shark bite on December 8th, 2020 on the island of Maui, from swab samples collected from the victim's surfboard 49 h after the bite incident. Our experimental results demonstrate that shark species can be accurately identified from transfer DNA recovered from bite impressions on surfboards and wetsuit neoprene. The successful use of our method in the real-world incident shows great potential for the practicality of this tool. We recommend DNA swabbing as a routine part of the forensic analysis of shark bites to help identify the species involved in human-shark interactions., (© 2021 American Academy of Forensic Sciences.)

The top challenges of adopting new methods to forensic DNA analysis in routine laboratories are often the capital investment and the expertise required to implement and validate such methods locally. In the case of next-generation sequencing, in the last decade, several specifically forensic commercial options became available, offering reliable and validated solutions. Despite this, the readily available expertise to analyze, interpret and understand such data is still perceived to be lagging behind. This review gives an introductory overview for the forensic scientists who are at the beginning of their journey with implementing next-generation sequencing locally and because most in the field do not have a bioinformatics background may find it difficult to navigate the new terms and analysis options available. The currently available open-source and commercial software for forensic sequencing data analysis are summarized here to provide an accessible starting point for those fairly new to the forensic application of massively parallel sequencing.

Strigiformes are affected by a substantial decline mainly caused by habitat loss and destruction, poaching, and trapping. Moreover, the increasing trend in bird trade and the growing interest in wild-caught rather than captive-bred birds are expected to encourage illegal trade. The biomolecular investigation represents a valuable tool to track illegal trade and to explore the genetic variability to preserving biodiversity. Microsatellite loci (STRs) are the most used markers to study genetic variability. Despite the availability of species-specific microsatellite loci in Strigiformes, a unique panel permitting the description of the genetic variability across species has not been identified yet. We tested 32 highly polymorphic microsatellite markers to evaluate the reliability of a unique microsatellite panel in different species of Strigiformes and its use for conservation and forensic purposes. We included in the study 84 individuals belonging to 28 parental groups and 11 species of Strigiformes. After screening polymorphic microsatellite loci, the description of genetic variability, and the kinship assessment, we characterized a final panel of 12 microsatellite loci able to identify individuals in 9 Strigiformes species. This STR panel might support the authorities in the forensic investigation for suspected smugglers and false parental claims; moreover, it can be useful to evaluate relatedness among individuals in captive-bred populations and to implement research projects finalized to the description of the genetic variability in wild populations.

Wet-lab based studies have exploited emerging single-cell technologies to address the challenges of interpreting forensic mixture evidence. However, little effort has been dedicated to developing a systematic approach to interpreting the single-cell profiles derived from the mixtures. This study is the first attempt to develop a comprehensive interpretation workflow in which single-cell profiles from mixtures are interpreted individually and holistically. In this approach, the genotypes from each cell are assessed, the number of contributors (NOC) of the single-cell profiles is estimated, followed by developing a consensus profile of each contributor, and finally the consensus profile(s) can be used for a DNA database search or comparing with known profiles to determine their potential sources. The potential of this single-cell interpretation workflow was assessed by simulation with various mixture scenarios and empirical allele drop-out and drop-in rates, the accuracies of estimating the NOC, the accuracies of recovering the true alleles by consensus, and the capabilities of deconvolving mixtures with related contributors. The results support that the single-cell based mixture interpretation can provide a precision that cannot beachieved with current standard CE-STR analyses. A new paradigm for mixture interpretation is available to enhance the interpretation of forensic genetic casework.

This study introduces a methodology for inferring the weight of the evidence (WoE) in the single nucleotide polymorphism (SNP)-typed DNA mixtures of forensic interest. First, we redefined some algebraic formulae to approach the semi-continuous calculation of likelihoods and likelihood ratios (LRs). To address the allelic dropouts, a peak height ratio index ("h," an index of heterozygous state plausibility) was incorporated into semi-continuous formulae to act as a proxy for the "split-drop" model of calculation. Second, the original ratio at which a person of interest (POI) has entered into the mixture was inferred by evaluating the DNA amounts conferred by unique genotypes to any possible permutation of any locus of the typing protocol (unique genotypes are genotypes that appear just once in the relevant permutation). We compared this expected ratio (MRex) to all the mixing ratios emerging at all other permutations of the mixture (MRobs) using several (1 - χ2) tests to evaluate the probability of each permutation to exist in the mixture according to quantitative criteria. At the level of each permutation state, we multiplied the (1 - χ2) value to the genotype frequencies and the h index. All the products of all the permutation states were finally summed to give a likelihood value that accounts for three independent properties of the mixtures. Owing to the (1 - χ2) index and the h index, this approach qualifies as a fully continuous methodology of LR calculation. We compared the MRs and LRs emerging from our methodology to those generated by the EuroForMix software ver. 3.0.3. When the true contributors were tested as POIs, our procedure generated highly discriminant LRs that, unlike EuroForMix, never overcame the corresponding single-source LRs. When false contributors were tested as POIs, we obtained a much lower LR value than that from EuroForMix. These two findings indicate that our computational method is more reliable and realistic than EuroForMix., Competing Interests: The authors have declared that no competing interests exist.

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.)

Rootless hair shafts are often considered unsuitable for STR genotyping due to the known high failure rate. The same samples can be reliably processed with mitochondrial sequencing. However, the minimal discriminatory power of widely implemented control region mitochondrial sequencing techniques limits its utility in some forensic casework. In this research, multiple variables were tested to provide information on rootless hair shaft sample genotyping success. Results showed external decontamination procedures decreased drop-in alleles but also greatly reduced profile recovery. The novel InnoXtract ™ chemistry was comparable to automated EZ1 DNA Investigator extraction. With thoroughly decontaminated hairs, InnoTyper ® 21 amplification generated random match probabilities higher than STR chemistry in 71.875% of samples and 18.75% of samples benefitted from the use of InnoTyper ® 21 amplification compared with estimated mtDNA profile rarity. Compared with the capillary electrophoresis-based amplification chemistries tested, the ForenSeq™ DNA Signature Prep chemistry paired with massively parallel sequencing was the most discriminatory amplification strategy tested., (© 2021 American Academy of Forensic Sciences.)

Casework evidence samples are likely to be placed under diverse and harsh environments as compared to quantified DNA samples including serial-diluted standard DNA samples. Internal validation of a novel STR kit using casework evidence sample, which is conducted according to various conditions such as DNA contamination and degradation, is crucial before being used as a forensic application. Therefore, this study aimed to elucidate the reliability of the Investigator® 24plex QS kit through DNA derived from casework evidence and to assess whether it is applicable to STR analysis together with PowerPlex® Fusion System and GlobalFiler™ PCR Amplification Kit. DNA was extracted from 189 casework evidence samples in a total of 77 cases. The mismatch of the allelic size of this kit through allelic sizing precision test, was suitable according to ENFSI guidelines. All heterozygous balance of the three kits were above 0.6 recommended value of ENFSI guideline. The number of allele drop-in was most frequent in the GlobalFiler™ PCR Amplification Kit. In addition, the number of allele drop-out was most frequent in the Investigator® 24plex QS kit. The cutoff concentration of DNA detected in three kits of one complete STR was approximately 45 pg/μL on average. Despite of several limitations, the Investigator® 24plex QS kit is considered to have the capability to be used for STR analysis of casework evidence samples., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Single-cell sequencing is a fast developing and very promising field; however, it is not commonly used in forensics. The main motivation behind introducing this technology into forensics is to improve mixture deconvolution, especially when a trace consists of the same cell type. Successful studies demonstrate the ability to analyze a mixture by separating single cells and obtaining CE-based STR profiles. This indicates a potential use of the method in other forensic investigations, like forensic DNA phenotyping, in which using mixed traces is not fully recommended. For this study, we collected single-source autopsy blood from which the white cells were first stained and later separated with the DEPArray™ N×T System. Groups of 20, 10, and 5 cells, as well as 20 single cells, were collected and submitted for DNA extraction. Libraries were prepared using the Ion AmpliSeq™ PhenoTrivium Panel, which includes both phenotype (HIrisPlex-S: eye, hair, and skin color) and ancestry-associated SNP-markers. Prior to sequencing, half of the single-cell-based libraries were additionally amplified and purified in order to improve the library concentrations. Ancestry and phenotype analysis resulted in nearly full consensus profiles resulting in correct predictions not only for the cells groups but also for the ten re-amplified single-cell libraries. Our results suggest that sequencing of single cells can be a promising tool used to deconvolute mixed traces submitted for forensic DNA phenotyping.

GlobalFiler is a new PCR amplification kit that includes 21 autosomal short tandem repeats and three sex-determining loci. In the present research, for the first time, the GlobalFiler kit was tested to analyze a sample of 500 unrelated individuals from 18 villages encompassing the entire area of Sardinia (Italy). We tested if the kit, which is a powerful tool in forensic studies, may also find application in the field of population genetics. In agreement with data from the literature on forensic parameters values, marker SE33 showed the highest degree of polymorphism, whereas TPOX was the least informative locus. Seventeen out of twenty-one autosomal markers included in the kit resulted highly polymorphic, and therefore Globalfiler turned out to be highly useful for forensic analysis in the Sardinian population. Moreover, our data suggest developing different STR databases in different populations, like Sardinians, to increase the statistical power of autosomal STR profiling. On the other hand, due to the presence of some very highly polymorphic markers, the efficiency of Globalfiler in detecting geographical variability is affected. Indeed, the differentiation previously observed between the Sardinian and Italian populations appeared greatly reduced and even the presence of genetic isolates, previously recorded when uniparental markers was not revealed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DNA methylation analysis is becoming increasingly useful in biomedical research and forensic practice. The discovery of differentially methylated sites (DMSs) that continuously change over an individual's lifetime has led to breakthroughs in molecular age estimation. Although semen samples are often used in forensic DNA analysis, previous epigenetic age prediction studies mainly focused on somatic cell types. Here, Infinium MethylationEPIC BeadChip arrays were applied to semen-derived DNA samples, which identified numerous novel DMSs moderately correlated with age. Validation of the ten most age-correlated novel DMSs and three previously known sites in an independent set of semen-derived DNA samples using targeted bisulfite massively parallel sequencing, confirmed age-correlation for nine new and three previously known markers. Prediction modelling revealed the best model for semen, based on 6 CpGs from newly identified genes SH2B2 , EXOC3 , IFITM2 , and GALR2 as well as the previously known FOLH1B gene, which predict age with a mean absolute error of 5.1 years in an independent test set. Further increases in the accuracy of age prediction from semen DNA will require technological progress to allow sensitive, simultaneous analysis of a much larger number of age correlated DMSs from the compromised DNA typical of forensic semen stains.

In cases involving identification of missing persons, mass disasters and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA profiling. Standard methods for extraction of DNA from such samples involve grinding of the bone and teeth samples. Here, we present an extremely efficient protocol for recovery of DNA from bone samples by a method of scrapping. The study was carried out on 25 samples and it was found that the quantity of DNA isolated by the scrapping method was up to 1.131 ng/µl with a success rate of 93% as compared to a much lower yield of 0.359 ng/µl DNA isolated with a success rate of 28% through the grinding method. The scrapping method of DNA extraction has been proven to be extremely useful in forensic examination of challenging samples that had multiple failures using the traditional grinding method., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest whatsoever and all the authors have read and approved the manuscript., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Kinship testing based on genetic relatedness is one of the major tasks in forensic genetics. Although short tandem repeats (STRs) are the "gold standard" biomarkers for relationship testing, microhaplotypes (MHs) have also emerged as viable options for kinship elucidation. In this work, the kinship testing efficiency of 54 highly polymorphic MHs was studied in two extended families consisting of parent-offspring, full siblings, grandparent-grandchildren, uncle/aunt-nephew/nieces, and first cousins. In addition, ten-thousand pairs of different degrees of relationships were simulated using various datasets including 54 MHs, 27 STRs plus 94 single nucleotide polymorphisms (SNPs) that were included in the ForenSeq DNA Signature Prep Kit (ForenSeq), 54 MHs plus loci in ForenSeq, and different subsets of 417-published MHs. The panels' system effectiveness in the kinship analysis were accessed by likelihood ratio distributions. The results showed that 54 MHs could be used in first-degree relationship testing with high reliability. The effectiveness of 54 MHs was slightly lower than ForenSeq but only by a narrow margin. Both 54 MHs and ForenSeq were not sufficient for distant relationship testing, and approximately 200 microhaplotypes with an average expected heterozygosity (He) = 0.79 were enough to determine second-degree relationships, but a panel of 417 MHs with an average He = 0.72 was not sufficient to first cousins testing according to the simulation analysis. In conclusion, 54 MHs could be used to serve as supplement markers for kinship testing; and well-established STR markers plus well-performing microhaplotype markers may become collective tools in forensic applications, though an enlarged pool of forensic markers is needed for distant relationship testing., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) has organized a second collaborative exercise on a simulated case of Disaster Victim Identification (DVI), with the participation of eighteen laboratories. The exercise focused on the analysis of a simulated plane crash case of medium-size resulting in 66 victims with varying degrees of fragmentation of the bodies (with commingled remains). As an additional difficulty, this second exercise included 21 related victims belonging to 6 families among the 66 missings to be identified. A total number of 228 post-mortem samples were represented with aSTR and mtDNA profiles, with a proportion of partial aSTR profiles simulating charred remains. To perform the exercise, participants were provided with aSTR and mtDNA data of 51 reference pedigrees -some of which deficient-including 128 donors for identification purposes. The exercise consisted firstly in the comparison of the post-mortem genetic profiles in order to re-associate fragmented remains to the same individual and secondly in the identification of the re-associated remains by comparing aSTR and mtDNA profiles with reference pedigrees using pre-established thresholds to report a positive identification. Regarding the results of the post-mortem samples re-associations, only a small number of discrepancies among participants were detected, all of which were from just a few labs. However, in the identification process by kinship analysis with family references, there were more discrepancies in comparison to the correct results. The identification results of single victims yielded fewer problems than the identification of multiple related victims within the same family groups. Several reasons for the discrepant results were detected: a) the identity/non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, b) some laboratories failed to use all family references to report the DNA match, c) In families with several related victims, some laboratories firstly identified some victims and then unnecessarily used their genetic information to identify the remaining victims within the family, d) some laboratories did not correctly use "prior odds" values for the Bayesian treatment of the episode for both post-mortem/post-mortem re-associations as well as the ante-mortem/post-mortem comparisons to evaluate the probability of identity. For some of the above reasons, certain laboratories failed to identify some victims. This simulated "DNA-led" identification exercise may help forensic genetic laboratories to gain experience and expertize for DVI or MPI in using genetic data and comparing their own results with the ones in this collaborative exercise., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The analysis of DNA methylation levels of specific CpG sites is one of the most promising molecular techniques to estimate an individual's age. Numerous studies were published recently presenting age estimation models based on DNA methylation patterns from blood samples, with only a few using saliva or buccal swabs. The aim of this study was to identify age-dependent methylation of 88 CpG sites in eight different marker regions (PDE4C, ELOVL2, ITGA2B, ASPA, EDARADD, SST, KLF14 and SLC12A5) in buccal swab samples. A total of 141 buccal swabs from individuals with age ranging from 21 to 69 years were split into a training set (n = 95) and a validation set (n = 46). Samples of the training set were analyzed by pyrosequencing and markers with best age correlation were identified. Stepwise linear regression analysis was performed resulting in an age estimation model including three of the examined CpG sites and showing a mean absolute deviation of estimated from chronological age of 5.11 years. To allow easy implementation into forensic laboratories without the need for pyrosequencing equipment, a multiplex minisequencing reaction was developed, including the same CpG sites previously identified by pyrosequencing. An adjusted age estimation model was evaluated with a mean absolute deviation of estimated from chronological age of 5.16 years. The independent validation set of 46 buccal swab samples was used to test model performances. Mean absolute deviation of estimated from chronological age was 5.33 years and 6.44 years for the pyrosequencing model and the minisequencing model, respectively. Comparison of the two methods showed a high concordance of results, both, qualitatively and quantitatively. In conclusion, buccal swabs offer a suitable alternative to blood samples for molecular age estimation with the additional advantage of being collected non-invasively. Furthermore we showed that minisequencing offers a cost-effective and easy-to-integrate alternative to pyrosequencing for the analysis of methylation status of individual CpG sites., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bayesian logistic regression is used to model the probability of DNA recovery following direct and secondary transfer and persistence over a 24 h period between deposition and sample collection. Sub-source level likelihood ratios provided the raw data for activity-level analysis. Probabilities of secondary transfer are typically low, and there are challenges with small data-sets with low numbers of positive observations. However, the persistence of DNA over time can be modelled by a single logistic regression for both direct and secondary transfer, except that the time since deposition must be compensated by an offset value for the latter. This simplifies the analysis. Probabilities are used to inform an activity-level Bayesian Network that takes account of alternative propositions e.g. time of assault and time of social activities. The model is extended in order to take account of multiple contacts between person of interest and 'victim'. Variables taken into account include probabilities of direct and secondary transfer, along with background DNA from unknown individuals. The logistic regression analysis is Bayesian - for each analysis, 4000 separate simulations were carried out. Quantile assignments enable calculation of a plausible range of probabilities and sensitivity analysis is used to describe the corresponding variation of LRs that occur when modelled by the Bayesian network. It is noted that there is need for consistent experimental design, and analysis, to facilitate inter-laboratory comparisons. Appropriate recommendations are made. The open-source program written in R-code ALTRaP (Activity Level, Transfer, Recovery and Persistence) enables analysis of complex multiple transfer propositions that are commonplace in cases-work e.g. between those who cohabit. A number of case examples are provided. ALTRaP can be used to replicate the results and can easily be modified to incorporate different sets of data and variables., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

A European-wide online survey was conducted to generate an overview on the state-of-the-art using massively parallel sequencing (MPS) platforms for forensic DNA analysis and DNA phenotyping among forensic practitioners in Europe. The survey was part of the dissemination activities of the "VISible Attributes through GEnomics - VISAGE" Horizon 2020 funded European research project [30], in preparation of a series of educational training activities. A total of 105 replies from 32 European countries representing participants from police, governmental, academic, and private laboratories providing professional services in the field of forensic genetics were included in the final analysis. Of these, 73% already own an MPS platform or are planning to acquire one within the next 1-2 years. One-third of the participants have already carried out MPS-based STR sequencing, identity, or ancestry SNP typing. A total of 23-40% of participants are planning to explore all FDP applications showing the overall very high interest in using MPS for the whole range of forensic MPS markers and applications. About 50% of the participants have previously gathered experience using forensic DNA phenotyping (FDP) markers based on conventional (i.e., not MPS-based) DNA typing methods. A total of 55% of the participants have attended training on the general use of MPS technology, but 36% have received no training whatsoever. Accordingly, 90% have expressed high or medium interest to attend training on the analysis and interpretation of DNA phenotyping data for predicting appearance, ancestry, and age. The results of our survey will provide valuable information for organizing relevant training workshops on all aspects of MPS-based DNA phenotyping for the forensic genetics scientific community.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.

Evaluating forensic biological evidence considering activity level propositions is becoming more prominent around the world. In such evaluations it is common to combine results from multiple items associated with the alleged activities. The results from these items may not be conditionally independent, depending on the mechanism of cell/DNA transfer being considered and it is important that the evaluation takes these dependencies into account. Part of this consideration is to incorporate our understanding of prevalent DNA and of background DNA on objects and people, and how activities can lead to common sources of unknown DNA being deposited on items. We demonstrate a framework for evaluation of DNA evidence in such a scenario using Object-Oriented Bayesian Networks and apply it to a motivating case from South Australia., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400 bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10 ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.

The prediction of human externally visible characteristics (EVCs) based solely on DNA information has become an established approach in forensic and anthropological genetics in recent years. While for a large set of EVCs, predictive models have already been established using multinomial logistic regression (MLR), the prediction performances of other possible classification methods have not been thoroughly investigated thus far. Motivated by the question to identify a potential classifier that outperforms these specific trait models, we conducted a systematic comparison between the widely used MLR and three popular machine learning (ML) classifiers, namely support vector machines (SVM), random forest (RF) and artificial neural networks (ANN), that have shown good performance outside EVC prediction. As examples, we used eye, hair and skin color categories as phenotypes and genotypes based on the previously established IrisPlex, HIrisPlex, and HIrisPlex-S DNA markers. We compared and assessed the performances of each of the four methods, complemented by detailed hyperparameter tuning that was applied to some of the methods in order to maximize their performance. Overall, we observed that all four classification methods showed rather similar performance, with no method being substantially superior to the others for any of the traits, although performances varied slightly across the different traits and more so across the trait categories. Hence, based on our findings, none of the ML methods applied here provide any advantage on appearance prediction, at least when it comes to the categorical pigmentation traits and the selected DNA markers used here., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

On the morning of October 19, 2004, an eight-year-old boy and a 56-year-old woman were stabbed to death on an open street in the city of Linköping, Sweden. The perpetrator left his DNA at the crime scene, and after 15 years of various investigation efforts, including more than 9000 interrogations and mass DNA screening of more than 6000 men, there were still no clues about the identity of the unknown murderer. The successful application of investigative genetic genealogy (IGG) in the US raised the interest for this tool within the Swedish Police Authority. After legal consultations it was decided that IGG could be applied in this double murder case as a pilot case study. From extensive DNA analysis, including whole-genome sequencing and genotype imputation, DNA data sets were established and searched within both GEDmatch and FamilyTree DNA genealogy databases. A number of fairly distant relatives were found from which family trees were created. The genealogy work resulted in two candidates, two brothers, one of whom matched the crime scene samples by routine STR profiling. The suspect confessed the murders at the initial police hearing and was later convicted of the murders. In this paper we describe the successful application of an emerging technology. We disclose details of the DNA analyses which, due to the poor quality and low quantity of the DNA, required reiterative sequencing and genotype imputation efforts. The successful application of IGG in this double murder case exemplifies its applicability not only in the US but also in Europe. The pressure is now high on the involved authorities to establish IGG as a tool for cold case criminal investigations and for missing person identifications. There is, however, a continuous need to accommodate legal, social and ethical aspects as well., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

When DNA profiles obtained from biological evidence at a crime scene fail to match suspects or anyone in the database, forensic DNA phenotyping, which is the prediction of externally visible characteristics, can facilitate a traced search for an unknown suspect by limiting the search range. Therefore, age, trait, or lifestyle predictors, as well as the predictor for colorations, have been researched in the forensic field. In the present study, for the development of a prediction model for BMI or obesity, we investigated several previously reported BMI- or obesity-associated genetic and epigenetic markers that included four CpGs (cg06500161, cg00574958, cg12593793, and cg10505902 of the ABCG1, CPT1A, LMNA, and PDE4DIP genes, respectively), and eight SNPs (rs12463617, rs1558902, rs591166, rs11030104, rs11671664, rs6545814, rs16858082, and rs574367 near the TMEM18, FTO, MC4R, BDNF, GIPR/QPCTL, ADCY3/RBJ, GNPDA2, and SEC16B genes, respectively) in 700 Koreans within the BMI ranging from 16.1 to 40.6 (27.6 ± 4.5) kg/m 2 . Linear regression analysis showed that DNA methylation of the four CpG sites explained 10.9% total variance in BMI, and the model constructed using age information, genetic score from eight SNPs, and DNA methylation at four CpG sites could account for 17.4% of BMI variance. Using data mining techniques, i.e., decision tree (Entropy and Gini), random forest, and bagging, a total of eight models with BMI 31 or 32 as a cutoff value were also constructed based on the data obtained from 490 training samples with age and sex as a covariate. Among them, a random forest model with a cutoff value of 31 showed the best performance with 63.3% accuracy and the AUC value of 0.682 in 210 test set samples. In the present study, we could replicate the previous finding that DNA methylation contributes more to BMI than do genetic factors. In addition, although the accuracy for the prediction of BMI was not high, our study is meaningful in respect of the ability to use a small number of markers to achieve similar prediction accuracy to that obtained from a model composed of more than a thousand markers, which adds support to continued research to identify a small set of predictive markers for practical application in the forensic field.

The shedder status of an individual may be important to consider in the context of DNA transfer, persistence and recovery and in Bayesian networks where a person's shedder status may have an impact on the outcome. In this study we compared two methods to determine shedder status: the handheld tube (HH) method and a fluorescent cell count (CC) method. A poor association was observed between the numbers of detected cells in a fingerprint using the CC method and the strength of the DNA result with the HH method. The 20 participants were classified into low (25%), medium (50%) and high (25%) shedders based on the HH method. While the low and high shedders showed a good consistency between the replicates, the medium shedders varied more and have to be considered more carefully as they may act as either a high or a low shedder in an event of DNA transfer., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The popularity of dogs as human companions explains why these pets regularly come into focus in forensic cases such as bite attacks or accidents. Canine evidence, e.g., dog hairs, can also act as a link between the victim and suspect in a crime case due to the close contact between dogs and their owners. In line with human DNA identification, dog individualization from crime scene evidence is mainly based on the analysis of short tandem repeat (STR) markers. However, when the DNA profile does not match a reference, additional information regarding the appearance of the dog may provide substantial intelligence value. Key features of the dog's appearance, such as the body size and coat colour are well-recognizable and easy to describe even to non-dog experts, including most investigating officers and eyewitnesses. Therefore, it is reasonable to complement eyewitnesses' testimonies with externally visible traits predicted from associated canine DNA samples. Here, the feasibility and suitability of canine DNA phenotyping is explored from scratch in the form of a proof of concept study. To predict the overall appearance of an unknown dog from its DNA as accurately as possible, the following six traits were chosen: (1) coat colour, (2) coat pattern, (3) coat structure, (4) body size, (5) ear shape, and (6) tail length. A total of 21 genetic markers known for high predicting values for these traits were selected from previously published datasets, comprising 15 SNPs and six INDELS. Three of them belonged to SINE insertions. The experiments were designed in three phases. In the first two stages, the performance of the markers was tested on DNA samples from dogs with well-documented physical characteristics from different breeds. The final blind test, including dogs with initially withheld appearance information, showed that the majority of the selected markers allowed to develop composite sketches, providing a realistic impression of the tested dogs. We regard this study as the first attempt to evaluate the possibilities and limitations of forensic canine DNA phenotyping.

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.

Police dogs are routinely deployed during criminal investigations under a variety of circumstances. In instances where police dogs are involved in apprehension of suspects, contact with a suspect may be observed or may occur out of the line of sight. The interactions between suspect and dog may include the dog biting the suspect, or the suspect touching or exuding bodily fluids onto the dog. In either form of contact, potentially valuable DNA may be left from the suspect on the dog. This paper describes a proof-of-concept study investigating collection of human DNA from the teeth and hair of dogs. It used controlled settings, where the human DNA sources were touch and saliva, and field cases, where the human DNA sources were unknown. The results of sample analysis to identify DNA short tandem repeats (STRs) from each of these scenarios are provided. They highlight the potential and importance of collecting trace DNA from police dogs who may have had contact with suspects during attempted apprehension.

Purpose: We investigated the recovery and extraction efficiency of DNA from three metal surfaces (brass, copper, steel) relevant to forensic casework, and plastic (control) using two different swabbing systems; Rayon and Isohelix™ swabs, with sterile water and isopropyl alcohol respectively, as the wetting solutions., Methods: Twenty nanograms of human genomic DNA were applied directly to Isohelix™ and Rayon swabs; and to the metal and plastic substrates. All substrates were left to dry for 24 h, followed by single wet swabbing and extraction with the DNA IQ™ System. DNA extracts were quantified using real time quantitative PCR assays with SYBR green chemistry., Results: DNA was extracted from directly seeded Isohelix™ swabs with a high efficiency of 98%, indicating effective DNA-release from the swab into the extraction buffer. In contrast, only 58% of input DNA was recovered from seeded Rayon swabs, indicating higher DNA retention by these swabs. Isohelix™ swabs recovered 32 - 53% of DNA from metal surfaces, whilst the Rayon swabs recovered 11-29%. DNA recovery was lowest from copper and highest from brass. Interestingly, Rayon swabs appeared to collect more DNA from the plastic surface than Isohelix™ swabs, however, due to the lower release of DNA from Rayon swabs they returned less DNA overall following extraction than Isohelix™ swabs., Conclusion: These results demonstrate that DNA samples deposited on metal surfaces can be more efficiently recovered using Isohelix™ swabs wetted with isopropyl alcohol than Rayon swabs wetted with sterile water, although recovery is affected by the substrate type.

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 both criminal cases and civil cases there is an increasing demand for the analysis of DNA mixtures involving relationships. The goal might be, for example, to identify the contributors to a DNA mixture where the unknown donors may be related, or to infer the relationship between individuals based on a DNA mixture. This paper applies a new approach to modelling and computation for DNA mixtures involving contributors with arbitrarily complex relationships to two real cases from the Spanish Forensic Police., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rapid and efficient processing of sexual assault evidence will accelerate forensic investigation and decrease casework backlogs. The standardized protocols currently used in forensic laboratories require the continued innovation to handle the increasing number and complexity of samples being submitted to forensic labs. Here, we present a new technique leveraging the integration of a bio-inspired oligosaccharide (i.e., Sialyl-Lewis X ) with magnetic beads that provides a rapid, inexpensive, and easy-to-use strategy that can potentially be adapted with current differential extraction practice in forensics labs. This platform (i) selectively captures sperm; (ii) is sensitive within the forensic cut-off; (iii) provides a cost effective solution that can be automated with existing laboratory platforms; and (iv) handles small volumes of sample (∼200 μL). This strategy can rapidly isolate sperm within 25 minutes of total processing that will prepare the extracted sample for downstream forensic analysis and ultimately help accelerate forensic investigation and reduce casework backlogs., (Copyright © 2020. Published by Elsevier B.V.)

The application of transcriptome analyses in forensic genetics has experienced tremendous growth and development in the past decade. The earliest studies and main applications were body fluid and tissue identification, using targeted RNA transcripts and a reverse transcription endpoint PCR method. A number of markers have been identified for the forensically most relevant body fluids and tissues and the method has been successfully used in casework. The introduction of Massively Parallel Sequencing (MPS) opened up new perspectives and opportunities to advance the field. Contrary to genomic DNA where two copies of an autosomal DNA segment are present in a cell, abundant RNA species are expressed in high copy numbers. Even whole transcriptome sequencing (RNA-Seq) of forensically relevant body fluids and of postmortem material was shown to be possible. This review gives an overview on forensic transcriptome analyses and applications. The methods cover whole transcriptome as well as targeted MPS approaches. High resolution forensic transcriptome analyses using MPS are being applied to body fluid/ tissue identification, determination of the age of stains and the age of the donor, the estimation of the post-mortem interval and to post mortem death investigations., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Messenger RNA (mRNA) profiling can identify body fluids present in a stain, yielding information on what activities could have taken place at a crime scene. To account for uncertainty in such identifications, recent work has focused on devising statistical models to allow for probabilistic statements on the presence of body fluids. A major hurdle for practical adoption is that evidentiary stains are likely to contain more than one body fluid and current models are ill-suited to analyse such mixtures. Here, we construct a likelihood ratio (LR) system that can handle mixtures, considering the hypotheses H 1 : the sample contains at least one of the body fluids of interest (and possibly other body fluids); H 2 : the sample contains none of the body fluids of interest (but possibly other body fluids). Thus, the LR-system outputs an LR-value for any combination of mRNA profile and set of body fluids of interest that are given as input. The calculation is based on an augmented dataset obtained by in silico mixing of real single body fluid mRNA profiles. These digital mixtures are used to construct a probabilistic classification method (a 'multi-label classifier'). The probabilities produced are subsequently used to calculate an LR, via calibration. We test a range of different classification methods from the field of machine learning, ways to preprocess the data and multi-label strategies for their performance on in silico mixed test data. Furthermore, we study their robustness to different assumptions on background levels of the body fluids. We find logistic regression works as well as more flexible classifiers, but shows higher robustness and better explainability. We test the system's performance on lab-generated mixture samples, and discuss practical usage in case work., (Copyright © 2020. Published by Elsevier B.V.)

mRNA markers provide a very promising method for the identification of human body fluids or tissues in the context of forensic investigations. Previous studies have shown that different body fluids can be distinguished from each other according to their specific mRNA biomarkers. In this study, we evaluated eight semen-specific mRNA markers (KLK3, NKX3-1, CKB, KLK2, PRAC1, SEMG1, TGM4, and SORD) that encompass 12 coding single nucleotide polymorphisms (cSNPs) to identify the semen contributor in a mixed stain. Five highly specific and sensitive mRNA markers for blood, menstrual blood, saliva, vaginal secretions, and skin were also incorporated into the PCR system as body fluid-positive controls. Reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR and SNaPshot mini-sequencing assays were established for the identification of semen-specific mRNA. The amplicon size ranged from 133 to 337 bp. The semen-specific system was examined against blood, menstrual blood, saliva, vaginal secretions, and skin swabs. The eight mRNA biomarkers were semen-specific and could be successfully typed in laboratory-generated mixtures composed of different body fluids supplemented with 1 ng of semen cDNA. This system possessed a high sensitivity that ranged from 1:10-1:100 for detecting trace amounts of semen in semen-containing body fluid mixtures. Additionally, our results demonstrated that the cSNPs polymorphisms included in the mRNA markers were concordant with genomic DNA (gDNA). Despite the presence of other body fluids, the system exhibited high sensitivity and specificity to the semen in the mixture. In future studies, we will add other cSNPs from the semen-specific genes using massively parallel sequencing to further improve our system., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Previous studies have demonstrated that microbial community succession during the decomposition of cadavers could be used to estimate the postmortem interval (PMI). However, the vast majority of the existing studies focused on exposed cadavers. In fact, burial cadavers are common scenarios for forensic investigations. In this study, the microbial communities from gravesoil, rectum and skin of burial SD rat cadavers during decomposition were characterized using 16S rRNA gene high-throughput sequencing. We predicted PMI based on the microbial community succession. Obvious differences in microbial community structures were observed between different stages of decomposition. Later decay stages had a lower alpha diversity compared to earlier decay stages. Significant linear relationships between similarities of the microbial communities and postmortem intervals were observed, manifesting regular succession over the course of decomposition. Furthermore, we combined random forest models with postmortem microbial features to predict PMI. The model explained 86.83%, 84.55% and 81.67% of the variation in the microbial community, with a mean absolute error of 1.82, 2.06 and 2.13 days within 60 days of decomposition for gravesoil, rectum and skin of burial cadavers, respectively. Overall, our results suggested that postmortem microbial community data could serve as a potential forensic tool to estimate accurate PMI of burial cadavers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Massively parallel sequencing (MPS) is a promising supplementary method for forensic casework in short tandem repeats (STRs) genotyping, owing to several advantageous features in comparison to traditional capillary electrophoresis (CE). However, the application of MPS in casework requires accessible datasets from the worldwide population to enrich the allele frequencies of sequence-based STR genotypes., Methods: In this study, we report the characterization of sequence-based allele frequencies of 58 STRs from a Tibetan population comprising 120 unrelated individuals using the ForenSeq™ DNA Signature Prep Kit. A concordance study evaluating MPS and CE allele data was performed to ensure that MPS is compatible with current CE-based forensic databases. The diversity of observed alleles, allele frequencies, and forensic parameters per locus by length (LB), sequence without flanking region (RSB), and sequence with flanking region (FSB) were analyzed and compared., Results: The concordance study demonstrated a concordance rate exceeding 99%. The combined random match probability (RMP) for the 26 A-STRs was 2.04 × 10 -29 , 1.93 × 10 -31 , and 9.56 × 10 -33 for LB, RSB, and FSB, respectively. Similar trends were observed in other forensic parameters resulting from the increase in the number of unique alleles available. A total of 111 and 113 unique haplotypes in the Y-STR loci were observed when using length-based and sequence-based alleles, respectively. In addition, we identified 35 novel alleles at 25 loci and 25 polymorphisms in the flanking regions at 17 STRs., Conclusions: Our data suggest that MPS- and CE-derived alleles are compatible. MPS-based analysis of the STR data substantially increased the allele diversity and improved the forensic parameters, which clearly demonstrated the advantages of MPS in comparison to CE. With more pooled data and larger-scale validation, MPS could play a valuable role in forensic genetics and might be an additional tool for routine casework., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Animal forensic genetics, where the focus is on non-human species, is broadly divided in two: domestic species and wildlife. When traces of a domestic species are relevant to a forensic investigation the question of species identification is less important, as the material comes from either a dog or a cat for instance, but more relevant may be the identification of the actual pet. Identification of a specific animal draws on similar methods to those used in human identification by using microsatellite markers. The use of cat short tandem repeats to link a cat hair to a particular cat paved the way for similar identification of dogs. Wildlife forensic science is becoming accepted as a recognised discipline. There is growing acceptance that the illegal trade in wildlife is having devasting effects on the numbers of iconic species. Loci on the mitochondrial genome are used to identify the most likely species present. Sequencing the whole locus may not be needed if specific bases can be targeted. There can be benefits of increased sensitivity using mitochondrial loci for species testing, but occasionally there is an issue if hybrids are present. The use of massively parallel DNA sequencing has a role in the identification of the ingredients of traditional medicines where studies found protected species to be present, and a potential role in future species assignments. Non-human animal forensic testing can play a key role in investigations provided that it is performed to the same standards as all other DNA profiling processes.

Scientific and technological progress in the field of forensic genetics is very useful in the resolution of criminal cases, but it entails the need for a deep ethical reflection, as the individual Fundamental Rights may be violated. This project aims to collect and compare the opinion of prisoners and prison officials on what characteristics the country's forensic database should have. In this context, 210 subjects were surveyed, 101 of them prisoners and the rest prison officials, from three different Spanish penitentiary centers. Among the results obtained, most prisoners and officials consider the national DNA database to be useful, and additionally, a 40% of the participants would support the integration of the profiles of the entire population. 64% considered it ethical to use the DNA profiles of the database as a tool for familial searching. Despite this, half of the respondents are concerned about the future uses of the DNA database. Integrating the opinion of these analyzed groups with other relevant judicial, scientific and ethical convictions, ensures the regulation between security and individual's Human Rights., (Copyright © 2020 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

Recovering DNA from modern incinerated bones can be challenging and may require alteration of routine DNA extraction protocols. It has been postulated that incinerated bones share some similarities with ancient bones, including fragmented DNA, surface contamination and highly mineralised structure, all of which can inhibit the successful recovery of genetic material. For this reason, ancient DNA extraction protocols are often used for incinerated modern samples; however, their effectiveness is still somewhat unclear. Much of this uncertainty exists around the demineralisation step of extraction, specifically the length of incubation and retention or removal of supernatant. As obtaining human samples for forensic research can be challenging, porcine models (Sus scrofa domesticus) are often used as substitutes. This study developed real time PCR assays for porcine nuclear DNA in order to investigate the effects of modified demineralisation protocols on DNA yield from femurs exposed to either short (60 min) or prolonged (120 min) burning. Gradient PCR results indicated 56 °C was the ideal amplification temperature for targeted amplicons, with melt curve analysis showing short and long amplicons corresponded to 80.3 °C and 83 °C peaks respectively. Results of altered extraction protocol showed a trend towards higher DNA yields from longer demineralisation periods however this was not significant. By comparison, retaining supernatant post-demineralisation resulted in significantly greater DNA yields compared to discarding it (P < 0.009). Although DNA content yield decreased with burn duration, the demineralisation treatment variations appeared to have the same effect for all burn lengths. These results suggest that for incinerated modern bone retaining the supernatant following demineralisation can dramatically increase DNA yield., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There are several indirect database searching approaches to identify the potential source of a forensic biological sample. These DNA-based approaches are familial searching, Y-STR database searching, and investigative genetic genealogy (IGG). The first two strategies use forensic DNA databases managed by the government, and the latter uses databases managed by private citizens or companies. Each of these search strategies relies on DNA testing to identify relatives of the donor of the crime scene sample, provided such profiles reside in the DNA database(s). All three approaches have been successfully used to identify the donor of biological evidence, which assisted in solving criminal cases or identifying unknown human remains. This paper describes and compares these approaches in terms of genotyping technologies, searching methods, database structures, searching efficiency, data quality, data security, and costs, and raises some potential privacy and legal considerations for further discussion by stakeholders and scientists. Y-STR database searching and IGG are advantageous since they are able to assist in more cases than familial searching readily identifying distant relatives. In contrast, familial searching can be performed more readily with existing laboratory systems. Every country or state may have its own unique economic, technical, cultural, and legal considerations and should decide the best approach(es) to fit those circumstances. Regardless of the approach, the ultimate goal should be the same: generate investigative leads and solve active and cold criminal cases to public safety, under stringent policies and security practices designed to protect the privacy of its citizenry., (© 2020 American Academy of Forensic Sciences.)