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Genome and transcriptome examinations have become more common due to Next-Generation Sequencing (NGS), which significantly increases throughput and depth coverage while reducing costs and time. Mitochondrial DNA (mtDNA) is often the marker of choice in degraded samples from archaeological and forensic contexts, as its higher number of copies can improve the success of the experiment. Among other sequencing strategies, amplicon-based NGS techniques are currently being used to obtain enough data to be analyzed. There are some pipelines designed for the analysis of ancient mtDNA samples and others for the analysis of amplicon data. However, these pipelines pose a challenge for non-expert users and cannot often address both ancient and forensic DNA particularities and amplicon-based sequencing simultaneously. To overcome these challenges, a user-friendly bioinformatic tool was developed to analyze the non-coding region of human mtDNA from degraded samples recovered in archaeological and forensic contexts. The tool can be easily modified to fit the specifications of other amplicon-based NGS experiments. A comparative analysis between two tools, MarkDuplicates from Picard and dedup parameter from fastp, both designed for duplicate removal was conducted. Additionally, various thresholds of PMDtools, a specialized tool designed for extracting reads affected by post-mortem damage, were used. Finally, the depth coverage of each amplicon was correlated with its level of damage. The results obtained indicated that, for removing duplicates, dedup is a better tool since retains more non-repeated reads, that are removed by MarkDuplicates. On the other hand, a PMDS = 1 in PMDtools was the threshold that allowed better differentiation between present-day and ancient samples, in terms of damage, without losing too many reads in the process. These two bioinformatic tools were added to a pipeline designed to obtain both haplotype and haplogroup of mtDNA. Furthermore, the pipeline presented in the present study generates information about the quality and possible contamination of the sample. This pipeline is designed to automatize mtDNA analysis, however, particularly for ancient samples, some manual analyses may be required to fully validate results since the amplicons that used to be more easily recovered were the ones that had fewer reads with damage, indicating that special care must be taken for poor recovered samples., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cuesta-Aguirre et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Forensic analysis heavily relies on DNA analysis techniques, notably autosomal Single Nucleotide Polymorphisms (SNPs), to expedite the identification of unknown suspects through genomic database searches. However, the uniqueness of an individual's genome sequence designates it as Personal Identifiable Information (PII), subjecting it to stringent privacy regulations that can impede data access and analysis, as well as restrict the parties allowed to handle the data. Homomorphic Encryption (HE) emerges as a promising solution, enabling the execution of complex functions on encrypted data without the need for decryption. HE not only permits the processing of PII as soon as it is collected and encrypted, such as at a crime scene, but also expands the potential for data processing by multiple entities and artificial intelligence services., Methods: This study introduces HE-based privacy-preserving methods for SNP DNA analysis, offering a means to compute kinship scores for a set of genome queries while meticulously preserving data privacy. We present three distinct approaches, including one unsupervised and two supervised methods, all of which demonstrated exceptional performance in the iDASH 2023 Track 1 competition., Results: Our HE-based methods can rapidly predict 400 kinship scores from an encrypted database containing 2000 entries within seconds, capitalizing on advanced technologies like Intel AVX vector extensions, Intel HEXL, and Microsoft SEAL HE libraries. Crucially, all three methods achieve remarkable accuracy levels (ranging from 96% to 100%), as evaluated by the auROC score metric, while maintaining robust 128-bit security. These findings underscore the transformative potential of HE in both safeguarding genomic data privacy and streamlining precise DNA analysis., Conclusions: Results demonstrate that HE-based solutions can be computationally practical to protect genomic privacy during screening of candidate matches for further genealogy analysis in Forensic Genetic Genealogy (FGG)., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In forensics, genetic human identification is generally achieved by nuclear STR DNA typing. However, forensic samples often yield DNA in exiguous quantity and low quality, impairing the generation of conclusive DNA profiles by STR typing. In such cases, mitochondrial DNA (mtDNA) can be used as an alternative solution in forensic human identification. The high copy number, small circular DNA, high mutation rate, maternal inheritance, and absence of recombination are mtDNA's key features in forensics. In this work, we review mtDNA characteristics, forensic applications, sequencing methodologies and present some relevant examples in the forensic science literature.

The case of the monozygotic (MZ) twin as a suspect demonstrates a practical problem in forensic casework. As the MZ twins are genetically identical, they share the same short tandem repeat (STR) profile. Many studies showed that older MZ twins have significant differences in overall content and genomic distribution of methylation between them. However, studies addressing the investigation of epigenetic MZ triplet differentiation in various forensic reference materials are lacking. Here, one triplet set of Egyptian MZ twins was used as an analog to a forensic case. The genome-wide methylation analysis was performed via the new Human Methylation EPIC BeadChip array. Following normalization methods, potential differentially methylated positions (DMPs) were discovered. This resulted in the detection of 24 potential DMPs in reference-type blood DNA and 11 potential DMPs in reference-type buccal DNA. Then, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to show the associated biological functions. Our findings revealed that the 35 potential DMPs were enriched in 283 significant GO terms. These terms are mainly enriched in the immune system. Overall, this study demonstrates the general feasibility of epigenetic MZ triplet differentiation in the forensic context and highlights that some potential DMPs identified in blood DNA were not informative in buccal DNA. This is due to various reasons, including the tissue specificity of DNA methylation., Competing Interests: Declaration of Competing Interest All authors declare that there are no any competing financial interests in relation to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The successful application of Forensic Investigative Genetic Genealogy (FIGG) to the identification of unidentified human remains and perpetrators of serious crime has led to a growing interest in its use internationally, including Australia. Routinely, FIGG has relied on the generation of high-density single nucleotide polymorphism (SNP) profiles from forensic samples using whole genome array (WGA) (∼650,000 or more SNPs) or whole genome sequencing (WGS) (millions of SNPs) for DNA segment-based comparisons in commercially available genealogy databases. To date, this approach has required DNA of a quality and quantity that is often not compatible with forensic samples. Furthermore, it requires the management of large data sets that include SNPs of medical relevance. The ForenSeq™ Kintelligence kit, comprising of 10,230 SNPs including 9867 for kinship association, was designed to overcome these challenges using a targeted amplicon sequencing-based method developed for low DNA inputs, inhibited and/or degraded forensic samples. To assess the ability of the ForenSeq™ Kintelligence workflow to correctly predict biological relationships, a comparative study comprising of 12 individuals from a family (with varying degrees of relatedness from 1st to 6th degree relatives) was undertaken using ForenSeq™ Kintelligence and a WGA approach using the Illumina Global Screening Array-24 version 3.0 Beadchip. All expected 1st, 2nd, 3rd, 4th and 5th degree relationships were correctly predicted using ForenSeq™ Kintelligence, while the expected 6th degree relationships were not detected. Given the (often) limited availability of forensic samples, findings from this study will assist Australian Law enforcement and other agencies considering the use of FIGG, to determine if the ForenSeq™ Kintelligence is suitable for existing workflows and casework sample types considered for FIGG., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflicts of interest., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Mitochondrial DNA (mtDNA) is an important genetic marker for degraded biological sample identification, maternal pedigree tracing, and population genetic structure study owing to its characteristics of high copy number, anti-degradable ring structure, and maternal inheritance. Whole mtDNA genome sequencing is an optimal method for the analysis of mtDNA polymorphism and heterogeneity because it allows for the comprehensive use of maternal genetic information. However, because of lacking quantitative evaluations for sequencing data, the scientific interpretation standards for mtDNA sequencing results of the previously used sequencing systems are often different, and false positive or false negative results are prone to occur when faced with the interference of nuclear genomic DNA, or the heterogeneities of mtDNA sequence and structure. In this study, we evaluated a novel mtDNA whole genome sequencing system using long fragment amplification strategy on the DNA nanoball (DNB) sequencing platform. This system demonstrated high sequencing quality and specific mtDNA sequencing efficiencies on positive control DNA and FTA bloodstain samples, as the average Q20 and Q30 values of the corresponding samples were 97.17 % and 91.93 %; 97.37 % and 92.48 %, respectively. The mean mapping percentages for the reference sequences of whole genome DNA (wgDNA), mtDNA, and nuclear genomic DNA (ngDNA) in the corresponding samples were 99.98 %, 99.97 %, 0.03 %, and 99.91 %, 99.40 %, 0.60 %; respectively. The average error calling rates for the bases A, C, G, and T of the whole mtDNA genome were 0.2519 %, 0.2550 %, 0.2906 %; and 0.2392 %, respectively. The efficacy of heteroplasmy identification was assessed using a set of theoretical sites with predetermined rates. These sites were created by combining the samples with known mtDNA haplotypes in certain proportions. The absolute errors between observed and theoretical heteroplasmy values were 89.59 %, 74.68 %, 50.20 %, 12.65 %, 8.31 %, and 4.85 %, while the theoretical heteroplasmy values were 5 %, 10 %, 20 %, 80 %, 90 %, and 95 %, respectively. The absolute error exhibited relative stability when the mtDNA sequencing depth exceeded 500×. Furthermore, the system sequencing efficiency was also confirmed among different kinds of samples, and these samples included natural samples (e.g., peripheral blood samples preserved on FTA cards for 2 and 11 years, and on filter paper for 6 and 9 years), degraded samples, sensitivity samples, samples derived from various bodily fluids, and maternal pedigree samples. In summary, the whole mtDNA genome sequencing system used for forensic identification demonstrated high performance in analyzing mtDNA sequence information, and showed significant prospects for forensic application and maternal genetic research., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Most of the sexual assault casework samples are of mixed sources. Forensic DNA laboratories are always in the requirement of a precise technique for the efficient separation of sperm and non-sperm DNA from mixed samples. Since the introduction of the differential extraction technique in 1985, it has seen significant advancements in the form of either chemicals used or modification of incubation times. Several automated and semi-automated techniques have also adopted the fundamentals of conventional differential extraction techniques. However, lengthy incubation, several manual steps, and carryover over non-sperm material in sperm fraction are some of the major limitations of this technique. Advanced cell separation techniques have shown huge promise in separating sperm cells from a mixture based on their size, shape, composition, and membrane structure and antigens present on sperm membranes. Such advanced techniques such as DEParray, ADE, FACS, LCM, HOT and their respective pros and cons have been discussed in this article. As current-day forensic techniques should be as per the line of Olympic slogan i.e., faster, higher, stronger, the advanced cell separation techniques show a huge potential to be implemented in the casework samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The inference of body fluids and tissues is critical in reconstructing crime scenes and inferring criminal behaviors. Nevertheless, present methods are incompatible with conventional DNA genotyping, and additional testing might result in excessive consumption of forensic scene materials. This study aims to investigate the feasibility of distinguishing common body fluids/tissues through the difference in mitochondrial DNA copy number (mtDNAcn). Four types of body fluids/tissues were analyzed in this study - hair, saliva, semen, and skeletal muscle. MtDNAcn was estimated by dividing the read counts of mitochondrial DNA to that of nuclear DNA (RR mt/nu ). Results indicated that there were significant differences in RR mt/nu between different body fluids/tissues. Specifically, hair samples exhibited the highest RR mt/nu (log 10 RR mt/nu : 4.3 ± 0.28), while semen samples showed the lowest RR mt/nu (log 10 RR mt/nu : -0.1 ± 0.28). RR mt/nu values for DNA samples without extraction were notably higher (approximately 2.9 times) than those obtained after extraction. However, no significant difference in RR mt/nu was observed between various age and gender groups. Hierarchical clustering and Kmeans clustering analyses showed that body fluids/tissues of the same type clustered closely to each other and could be inferred with high accuracy. In conclusion, this study demonstrated that the simultaneous detection of nuclear and mitochondrial DNA made it possible to perform conventional DNA analyses and body fluid/tissue inference at the same time, thus killing two birds with one stone. Furthermore, mtDNAcn has the potential to serve as a novel and promising biomarker for the identification of body fluids/tissues., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed., (© 2024. The Author(s).)

Considering activity level propositions in the evaluation of forensic biology findings is becoming more common place. There are increasing numbers of publications demonstrating different transfer mechanisms that can occur under a variety of circumstances. Some of these publications have shown the possibility of DNA transfer from site to site on an exhibit, for instance as a result of packaging and transport. If such a possibility exists, and the case circumstances are such that the area on an exhibit where DNA is present or absent is an observation that is an important diagnostic characteristic given the propositions, then site to site transfer should be taken into account during the evaluation of observations. In this work we demonstrate the ways in which site to site transfer can be built into Bayesian networks when carrying out activity level evaluations of forensic biology findings. We explore the effects of considering qualitative vs quantitative categorisation of DNA results. We also show the importance of taking into account multiple individual's DNA being transferred (such as unknown or wearer DNA), even if the main focus of the evaluation is the activity of one individual., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace's composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 - 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively., Competing Interests: Declaration of Competing Interest All authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Great apes are a global conservation concern, with anthropogenic pressures threatening their survival. Genetic analysis can be used to assess the effects of reduced population sizes and the effectiveness of conservation measures. In humans, autosomal short tandem repeats (aSTRs) are widely used in population genetics and for forensic individual identification and kinship testing. Traditionally, genotyping is length-based via capillary electrophoresis (CE), but there is an increasing move to direct analysis by massively parallel sequencing (MPS). An example is the ForenSeq DNA Signature Prep Kit, which amplifies multiple loci including 27 aSTRs, prior to sequencing via Illumina technology. Here we assess the applicability of this human-based kit in African great apes. We ask whether cross-species genotyping of the orthologs of these loci can provide both individual and (sub)species identification., Results: The ForenSeq kit was used to amplify and sequence aSTRs in 52 individuals (14 chimpanzees; 4 bonobos; 16 western lowland, 6 eastern lowland, and 12 mountain gorillas). The orthologs of 24/27 human aSTRs amplified across species, and a core set of thirteen loci could be genotyped in all individuals. Genotypes were individually and (sub)species identifying. Both allelic diversity and the power to discriminate (sub)species were greater when considering STR sequences rather than allele lengths. Comparing human and African great-ape STR sequences with an orangutan outgroup showed general conservation of repeat types and allele size ranges. Variation in repeat array structures and a weak relationship with the known phylogeny suggests stochastic origins of mutations giving rise to diverse imperfect repeat arrays. Interruptions within long repeat arrays in African great apes do not appear to reduce allelic diversity., Conclusions: Orthologs of most human aSTRs in the ForenSeq DNA Signature Prep Kit can be analysed in African great apes. Primer redesign would reduce observed variability in amplification across some loci. MPS of the orthologs of human loci provides better resolution for both individual and (sub)species identification in great apes than standard CE-based approaches, and has the further advantage that there is no need to limit the number and size ranges of analysed loci., (© 2024. The Author(s).)

Background : Forensic investigative genetic genealogy (FIGG) has developed rapidly in recent years and is considered a novel tool for crime investigation. However, crime scene samples are often of low quality and quantity and are challenging to analyze. Deciding which approach should be used for kinship inference in forensic practice remains a troubling problem for investigators. Methods : In this study, we selected four popular approaches-KING, IBS, TRUFFLE, and GERMLINE-comprising one method of moment (MoM) estimator and three identical by descent (IBD) segment-based tools and compared their performance at varying numbers of SNPs and levels of genotyping errors using both simulated and real family data. We also explored the possibility of making robust kinship inferences for samples with ultra-high genotyping errors by integrating MoM and the IBD segment-based methods. Results : The results showed that decreasing the number of SNPs had little effect on kinship inference when no fewer than 164 K SNPs were used for all four approaches. However, as the number decreased further, decreased efficiency was observed for the three IBD segment-based methods. Genotyping errors also had a significant effect on kinship inference, especially when they exceeded 1%. In contrast, MoM was much more robust to genotyping errors. Furthermore, the combination of the MoM and the IBD segment-based methods showed a higher overall accuracy, indicating its potential to improve the tolerance to genotyping errors. Conclusions : In conclusion, this study shows that different approaches have unique characteristics and should be selected for different scenarios. More importantly, the integration of the MoM and the IBD segment-based methods can improve the robustness of kinship inference and has great potential for applications in forensic practice.

Background: In the domain of forensic science, the application of kinship identification and mixture deconvolution techniques are of critical importance, providing robust scientific evidence for the resolution of complex cases. Microhaplotypes, as the emerging class of genetic markers, have been widely studied in forensics due to their high polymorphisms and excellent stability., Results and Discussion: In this research, a novel and high-efficient panel integrating 33 microhaplotype loci along with a sex-determining locus was developed by the next generation sequencing technology. In addition, we also assessed its forensic utility and delved into its capacity for kinship analysis and mixture deconvolution. The average effective number of alleles (Ae) of the 33 microhaplotype loci in the Guizhou Han population was 6.06, and the Ae values of 30 loci were greater than 5. The cumulative power of discrimination and cumulative power of exclusion values of the novel panel in the Guizhou Han population were 1-5.6 × 10 - 43 and 1-1.6 × 10 - 15 , respectively. In the simulated kinship analysis, the panel could effectively distinguish between parent-child, full-sibling, half-sibling, grandfather-grandson, aunt-nephew and unrelated individuals, but uncertainty rates clearly increased when distinguishing between first cousins and unrelated individuals. For the mixtures, the novel panel had demonstrated excellent performance in estimating the number of contributors of mixtures with 1 to 5 contributors in combination with the machine learning methods., Conclusions: In summary, we have developed a small and high-efficient panel for forensic genetics, which could provide novel insights into forensic complex kinships testing and mixture deconvolution., (© 2024. The Author(s).)

Modern forensic DNA quantitation assays provide information on the suitability of a DNA extract for a particular type of analysis, on the amount of sample to put into the analysis in order to yield an optimal (or best possible) result, and on the requirement for optional steps to improve the analysis. To achieve a high sensitivity and specificity, these assays are based on quantitative PCR (qPCR) and analyze target DNA loci that are present in multiple copies distributed across the genome. These target loci allow the determination of the amount of DNA, the degree of DNA degradation, and the proportion of DNA from male contributors. In addition, internal control DNA of a known amount is analyzed in order to inform about the presence of PCR inhibitors. These assays are nowadays provided as commercial kits that have been technically validated and are compatible with common qPCR instruments. In this review, the principles of forensic qPCR assays will be explained, followed by information on the nature of DNA loci targeted by modern forensic qPCR assays. Finally, we critically draw attention to the current trend of manufacturers not to disclose the exact nature of the target loci of their commercial kits.

Identical twins are also called monozygotic twins which originate from the same zygote that possesses the same genetic make-up. To discriminate between identical monozygotic twins, short tandem repeats has not been found effective, therefore, various techniques, including next-generation sequencing (NGS), are applied. Monozygotic twins can be identified through germ line genomes, through speech using deep learning networks, and through epigenetic analysis. Fingerprint analysis has also been used to distinguish between identical twins, as human beings have unique fingerprints. Two distinct levels of fingerprint are used to distinguish between monozygotic twins based upon the differences in the minutiae points. Examination of the methylation pattern of the genome has an enormous potential to differentiate between identical twins, as the methylation of DNA occurs uniquely to each individual. This article offers an insight into the latest methods and techniques used for the differentiation between the identical twins., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

The identification of tissue-specific differentially methylated regions has significantly contributed to the field of forensic genetics, particularly in body fluid identification crucial for linking evidence to crimes. Among the various approaches to analyzing DNA methylation, the SNaPshot assay has been popularly studied in numerous researches. However, there is a growing interest in exploring alternative methods such as the use of massively parallel sequencing (MPS), which can process a large number of samples simultaneously. This study compares SNaPshot and MPS multiplex assays using nine cytosine-phosphate-guanine markers for body fluid identification. As a result of analyzing 112 samples, including blood, saliva, vaginal fluid, menstrual blood, and semen, both methods demonstrated high sensitivity and specificity, indicating their reliability in forensic investigations. A total of 92.0% samples were correctly identified by both methods. Although both methods accurately identified all blood, saliva, and semen samples, some vaginal fluid samples showed unexpected methylation signals at nontarget loci in addition to the target loci. In the case of menstrual blood samples, due to their complexity, independent typing criteria were applied, and successful menstrual blood typing was possible, whereas a few samples showed profiles similar to vaginal fluid. The MPS method worked better in vaginal fluid samples, and the SNaPshot method performed better in menstrual blood samples. This study offers valuable insights into body fluid identification based on the characteristics of the SNaPshot and MPS methods, which may help in more efficient forensic applications., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

Combating wildlife crimes in South Africa requires accurate identification of traded species and their products. Diagnostic morphological characteristics needed to identify species are often lost when specimens are processed and customs officials lack the expertise to identify species. As a potential solution, DNA barcoding can be used to identify morphologically indistinguishable specimens in forensic cases. However, barcoding is hindered by the reliance on comprehensive, validated DNA barcode reference databases, which are currently limited. To overcome this limitation, we constructed a barcode library of cytochrome c oxidase subunit 1 and cytochrome b sequences for threatened and protected mammals exploited in southern Africa. Additionally, we included closely related or morphologically similar species and assessed the database's ability to identify species accurately. Published southern African sequences were incorporated to estimate intraspecific and interspecific variation. Neighbor-joining trees successfully discriminated 94%-95% of the taxa. However, some widespread species exhibited high intraspecific distances (>2%), suggesting geographic sub-structuring or cryptic speciation. Lack of reliable published data prevented the unambiguous discrimination of certain species. This study highlights the efficacy of DNA barcoding in species identification, particularly for forensic applications. It also highlights the need for a taxonomic re-evaluation of certain widespread species and challenging genera., Competing Interests: The authors declare there are no competing interests.

In forensic investigations, semen samples are a common form of biological evidence, especially in cases involving sexual assault. Therefore, accurately estimating the age of an individual is crucial in criminal cases. This study presents a novel age estimation model based on semen-specific CpG methylation patterns. A multiplex panel was developed, consisting of 12 CpG sites (PARP14, C5orf25, cg23488376, MXRA5, PFKFB3, DLL1, NOX4, cg12837463, TTC7B, KCNA7, NKX2-1, and SYNE4), which exhibit strong correlations with age. Additionally, this study investigates the resilience of these methylation markers under simulated environmental challenges. We collected ejaculate samples from a diverse cohort of 115 male individuals, aged 20-71 years, who underwent deoxyribonucleic acid extraction and bisulfite conversion. Methylation levels of the selected CpG sites were assessed using a SNaPshot assay, which revealed significant correlations with chronological age. We developed and validated two robust age estimation models through stepwise and enter regression analyses, achieving reliable accuracy with mean absolute errors ranging from 3.81 to 4.1 years. Additionally, the study also investigated the robustness of semen stains under diverse environmental conditions, including fabric type, washing, hematin exposure, and UV-C light. The selected methylation markers demonstrated remarkable resilience despite the challenges posed by washing procedures and environmental exposure, confirming their potential for age estimation in forensic genetics. This research presents successful age estimation models, emphasizing the strong correlations between methylation levels and chronological age. The proposed methodology's accuracy is affirmed through model validation on an independent test set, while also highlighting the resilience of semen stains on fabrics under varying storage and washing conditions., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

In forensic investigations, identifying the type of body fluid allows for the interpretation of biological evidence at the activity level. Over the past two decades, significant research efforts have focused on developing molecular methods for this purpose. MicroRNAs (miRNAs) hold great promise due to their tissue-specific expression, abundance, lack of splice variants, and relative stability. Although initial findings are promising, achieving consistent results across studies is still challenging, underscoring the necessity for both original and replication studies. To address this, we selected 18 miRNA candidates and tested them on 6 body fluids commonly encountered in forensic cases: peripheral blood, menstrual blood, saliva, semen, vaginal secretion, and skin. Using reverse transcription quantitative PCR analysis, we confirmed eight miRNA candidates (miR-144-3p, miR-451a, miR-205-5p, miR-214-3p, miR-888-5p, miR-891a-5p, miR-193b-3p, miR-1260b) with high tissue specificity and four (miR-203a-3p, miR-141-3p, miR-200b-3p, miR-4286) with lesser discrimination ability but still contributing to body fluid differentiation. Through principal component analysis and hierarchical clustering, the set of 12 miRNAs successfully distinguished all body fluids, including the challenging discrimination of blood from menstrual blood and saliva from vaginal secretion. In conclusion, our results provide additional data supporting the use of a small set of miRNAs for predicting common body fluids in forensic contexts. Large population data need to be gathered to develop a body fluid prediction model and assess its accuracy., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

The introduction of PCR into forensic science and the rapid increases in the sensitivity, specificity and discrimination power of DNA profiling that followed have been fundamental in shaping the field of forensic biology. Despite these developments, the challenges associated with the DNA profiling of trace, inhibited and degraded samples remain. Thus, any improvement to the performance of sub-optimal samples in DNA profiling would be of great value to the forensic community. The potential exists to optimise the PCR performance of samples by altering the cycling conditions used. If the effects of changing cycling conditions upon the quality of a DNA profile can be well understood, then the PCR process can be manipulated to achieve a specific goal. This work is a proof-of-concept study for the development of a smart PCR system, the theoretical foundations of which are outlined in part 1 of this publication. The first steps needed to demonstrate the performance of our smart PCR goal involved the manual alteration of cycling conditions and assessment of the DNA profiles produced. In this study, the timing and temperature of the denaturation and annealing stages of the PCR were manually altered to achieve the goal of reducing PCR runtime while maintaining an acceptable quality and quantity of DNA product. A real-time feedback system was also trialled using an STR PCR and qPCR reaction mix, and the DNA profiles generated were compared to profiles produced using the standard STR PCR kits. The aim of this work was to leverage machine learning to enable real-time adjustments during a PCR, allowing optimisation of cycling conditions towards predefined user goals. A set of parameters was found that yielded similar results to the standard endpoint PCR methodology but was completed 30 min faster. The development of an intelligent system would have significant implications for the various biological disciplines that are reliant on PCR technology.

In forensic genetics, utilizing massively parallel sequencing (MPS) to analyze short tandem repeats (STRs) has demonstrated several advantages compared to conventional capillary electrophoresis (CE). Due to the current technical limitations, although flanking region polymorphisms had been mentioned in several previous studies, most studies focused on the core repeat regions of STRs or the variations in the adjacent flanking regions. In this study, we developed an MPS system consisting of two sets of multiplex PCR systems to detect not only the STR core repeat regions but also to observe variants located at relatively distant positions in the flanking regions. The system contained 42 commonly used forensic STRs, including 21 autosomal STRs (A-STRs) and 21 Y-chromosomal STRs (Y-STRs), and a total of 350 male individuals from a Chinese Han population were genotyped. The length and sequence variants per locus were tallied and categorized based on length (length-based, LB), sequence without flanking region (core repeat regions sequence-based, RSB), and sequence with flanking region (core repeat and flanking regions sequence-based, FSB), respectively. Allele frequencies, Y-haplotype frequencies, and forensic parameters were calculated based on LB, RSB, and FSB, respectively, to evaluate the improvement in discrimination power, heterozygosity, and effectiveness of forensic systems. The results suggested the sequence variations have more influence on A-STRs and could improve the identification ability of MPS-STR genotyping. Concordance between MPS and CE methods was confirmed by using commercial CE-based STR kits. The impact of flanking region variations on STR genotype analysis and potential factors contributing to discordances were discussed. A total of 58 variations in the flanking regions (53 SNPs/SNVs and 5 InDels) were observed and most variations (48/58) were distributed in A-STRs. In summary, this study delved deeper into the genetic information of forensic commonly used STR and advanced the application of massively parallel sequencing in forensic genetics., (© 2024. The Author(s).)

The DNA intelligence tool, DNA methylation-based age prediction, can help identify disaster victims and suspects in criminal investigations. In this study, we developed a costal cartilage-based age prediction tool that uses massive parallel sequencing (MPS) of age-associated DNA methylation markers. Costal cartilage samples were obtained from 85 deceased Koreans, aged between 26 and 89 years. An MPS library was prepared using two rounds of multiplex polymerase chain reaction of nine genes (TMEM51, MIR29B2CHG, EDARADD, FHL2, TRIM59, ELOVL2, KLF14, ASPA, and PDE4C). The DNA methylation status of 45 CpG sites was determined and used to train an age prediction model via stepwise regression analysis. Nine CpGs in MIR29B2CHG, FHL2, TRIM59, ELOVL2, KLF14, and ASPA were selected for regression model construction. A leave-one-out cross-validation analysis revealed the high performance of the age prediction model, with a mean absolute error (MAE) and root mean square error of 4.97 and 6.43 years, respectively. Additionally, our model showed good performance with a MAE of 6.06 years in the analysis of data of 181 costal cartilage samples collected from Europeans. Our model effectively estimates the age of deceased individuals using costal cartilage samples; therefore, it can be a valuable forensic tool for disaster victim and missing person investigation., (© 2024 American Academy of Forensic Sciences.)

Closely linked groups of markers on the X chromosome are very useful for testing complex kinship relationships involving X-STR transmission. The Argus X-12 kit, a unique commercially available kit, can obtain haplotypes of 4 linkage groups (LGs) consisting of 3 markers. Although many population data have been reported for forensic purposes, differences in discrimination ability exist between LG1 and LG2, 3, and 4 in East Asian populations, and the data of this kit would become more useful if the discrimination ability of the latter groups were increased. Therefore, for matches found using this kit for some linkage group data, then to increase the identification ability, we additionally introduced 13 X-STR loci and established a method allowing comparison using data from 25 loci. The 13X-STRs add two locus data to each of LG2, 3, and 4, and also add two closely linked group (CLG) data between LG2 and 3 and LG3 and 4 in one multiplex PCR. Assessment of this method for a Malay population for which data by Argus X-12 had already been reported showed that the frequencies of distinct haplotypes in LG2, 3, and 4 were increased by 33.0-42.6 %, and frequencies of unique haplotypes increased by 45.4-59.2 %. The respective haplotype diversity values of the additional 3-locus and 4-locus CLGs were 0.9838 and 0.9939, which helps to improve discriminatory power and to predict recombination locations on the X chromosome. Although we have been testing these loci with Japanese subjects, this system would also be useful for the Malay population., 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. Published by Elsevier B.V.)

DNA mixtures are a common sample type in forensic genetics, and we typically assume that contributors to the mixture are unrelated when calculating the likelihood ratio (LR). However, scenarios involving mixtures with related contributors, such as in family murder or incest cases, can also be encountered. Compared to the mixtures with unrelated contributors, the kinship within the mixture would bring additional challenges for the inference of the number of contributors (NOC) and the construction of probabilistic genotyping models. To evaluate the influence of potential kinship on the individual identification of the person of interest (POI), we conducted simulations of two-person (2 P) and three-person (3 P) DNA mixtures containing unrelated or related contributors (parent-child, full-sibling, and uncle-nephew) at different mixing ratios (for 2 P: 1:1, 4:1, 9:1, and 19:1; for 3 P: 1:1:1, 2:1:1, 5:4:1, and 10:5:1), and performed massively parallel sequencing (MPS) using MGIEasy Signature Identification Library Prep Kit on MGI platform. In addition, in silico simulations of mixtures with unrelated and related contributors were also performed. In this study, we evaluated 1): the MPS performance; 2) the influence of multiple genetic markers on determining the presence of related contributors and inferring the NOC within the mixture; 3) the probability distribution of MAC (maximum allele count) and TAC (total allele count) based on in silico mixture profiles; 4) trends in LR values with and without considering kinship in mixtures with related and unrelated contributors; 5) trends in LR values with length- and sequence-based STR genotypes. Results indicated that multiple numbers and types of genetic markers positively influenced kinship and NOC inference in a mixture. The LR values of POI were strongly dependent on the mixing ratio. Non- and correct-kinship hypotheses essentially did not affect the individual identification of the major POI; the correct kinship hypothesis yielded more conservative LR values; the incorrect kinship hypothesis did not necessarily lead to the failure of POI individual identification. However, it is noteworthy that these considerations could lead to uncertain outcomes in the identification of minor contributors. Compared to length-based STR genotyping, using sequence-based STR genotype increases the individual identification power of the POI, concurrently improving the accuracy of mixing ratio inference using EuroForMix. In conclusion, the MGIEasy Signature Identification Library Prep kit demonstrated robust individual identification power, which is a viable MPS panel for forensic DNA mixture interpretations, whether involving unrelated or related contributors., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

During an investigation using Forensic Investigative Genetic Genealogy, which is a novel approach for solving violent crimes and identifying human remains, reference testing-when law enforcement requests a DNA sample from a person in a partially constructed family tree-is sometimes used when an investigation has stalled. Because the people considered for a reference test have not opted in to allow law enforcement to use their DNA profile in this way, reference testing is viewed by many as an invasion of privacy and by some as unethical. We generalize an existing mathematical optimization model of the genealogy process by incorporating the option of reference testing. Using simulated versions of 17 DNA Doe Project cases, we find that reference testing can solve cases more quickly (although many reference tests are required to substantially hasten the investigative process), but only rarely (<1%) solves cases that cannot otherwise be solved. Through a mixture of mathematical and computational analysis, we find that the most desirable people to test are at the bottom of a path descending from an ancestral couple that is most likely to be related to the target. We also characterize the rare cases where reference testing is necessary for solving the case: when there is only one descending path from an ancestral couple, which precludes the possibility of identifying an intersection (e.g., marriage) between two descendants of two different ancestral couples., (© 2024 American Academy of Forensic Sciences.)

This study explored secondary DNA transfer involving saliva, a body fluid often encountered in forensic investigations. Various factors were examined to investigate their potential impact on the transfer of DNA from saliva stains deposited onto common types of fabric (cotton, nylon, and towel). We examined varying types of saliva moisture (wet, dry, and rehydrated) and different types of contact (controlled pressure and active/friction pressure) to quantitatively evaluate how such variables could impact transfer and possible conclusions surrounding saliva-derived DNA deposits. The transfer of DNA was generally least pronounced with more absorbent primary fabrics (cotton and towel materials) while a less absorbent primary fabric (nylon) exhibited a greater propensity for DNA transfer. There were significantly higher amounts of transferred DNA (p < 0.05) observed in wet saliva samples compared to dry and rehydrated saliva samples. Further, the use of active pressure (friction) appeared to result in more DNA transfer overall as compared to controlled pressure contact. Experiments conducted with wet saliva and active pressure (friction) demonstrated the highest likelihood of transfer, with the primary nylon and secondary towel fabric combination demonstrating the greatest average transfer percentage of 94.74 %. The variables explored in this study presented multiple combinations wherein a sufficient amount of DNA (≥ 240 pg total) was transferred to the secondary fabric, making it potentially suitable for STR-PCR amplification in our laboratory. The findings from this study indicate that the type of primary fabric receiving the saliva deposit, the type of saliva moisture, the type of secondary fabric and its moisture type, and the type of contact all have the potential to affect the quantity of DNA transferred and recovered. This study provides empirical data on the ease, and to what extent, DNA from saliva transfers between fabrics and aids DNA activity level evaluations. The significance of this research lies in its contribution to expanding our current understanding of DNA transfer involving saliva within forensic science and criminal investigations., 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 B.V. All rights reserved.)

Investigative genetic genealogy (IGG) has emerged as a highly effective tool for tying a forensic DNA sample to an identity. While much of the attention paid to IGG has focused on cases where the DNA is from an unknown suspect, IGG has also been used to help close hundreds of unidentified human remains (UHR) cases. Genome-wide single-nucleotide polymorphism (SNP) genotype data can be obtained from forensic samples using microarray genotyping or whole-genome sequencing (WGS) with protocols optimized for degraded DNA. After bioinformatic processing, the SNP data can be uploaded to public GG databases that allow law enforcement usage, where it can be compared with other users' data to find distant relatives. A genetic genealogist can then build the family trees of the relatives to narrow down the identity of the source of the forensic DNA sample. To date, 367 UHR identifications using IGG have been publicly announced. The same IGG techniques developed and refined for UHR cases have significant potential for disaster victim identification, where DNA is often extremely compromised, and close family references may not be available. This paper reviews the laboratory, bioinformatic, and genealogical techniques used in IGG for UHR cases and presents three case studies that demonstrate how IGG is assisting with remains identification., (© 2024 American Academy of Forensic Sciences.)

Recently, RNA markers have been used to identify tissue origins of different kinds of body fluids. Herein, circRNA and miRNA markers were carried out to examine the presence or absence of peripheral blood (PB) in bloodstained samples exposed to different external environmental conditions, which mimicked PB samples left at the crime scenes. PB samples were placed on sterile swabs and then exposed to different high temperatures (37°C, 55°C and 95°C) and ultraviolet light irradiation for 0 d, 0.5 d, 1 d, 3 d, and 7 d, ultra-low and low temperatures (-80°C, -20°C, and 4°C) for 30 d, 180 d and 365 d and different kinds of disinfectants. Total RNA was extracted from bloodstained samples under the above different conditions, and the expressions of target RNAs (including miR16-5p, miR451a, circ0000095, and two reference genes RNU6b and 18 S rRNA) were detected by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. Results showed that these selected RNA markers could be successfully measured at all observation points with their unique degradation rates, which exhibited relative stability in degraded bloodstained samples exposed to different environmental conditions. This study provides insights into the applications of these studied miRNA and circRNA markers in forensic science., 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 B.V. All rights reserved.)

Devices of nanopore sequencing can be highly portable and of low cost. Thus, nanopore sequencing is promising in in-field forensic applications. Previous investigations have demonstrated that nanopore sequencing is feasible for genotyping forensic short tandem repeats (STRs) by using sequencers of Oxford Nanopore Technologies. Recently, Qitan Technology launched a new portable nanopore sequencer and became the second supplier in the world. Here, for the first time, we assess the QNome (QNome-3841) for its accuracy in nanopore sequencing of STRs and compare with MinION (MinION Mk1B). We profile 54 STRs of 21 unrelated individuals and 2800M standard DNA. The overall accuracy for diploid STRs and haploid STRs were 53.5% (378 of 706) and 82.7% (134 of 162), respectively, by using QNome. The accuracies were remarkably lower than those of MinION (diploid STRs, 84.5%; haploid, 90.7%), with a similar amount of sequencing data and identical bioinformatics analysis. Although it was not reliable for diploid STRs typing by using QNome, the haploid STRs were consistently correctly typed. The majority of errors (58.8%) in QNome-based STR typing were one-repeat deviations of repeat units in the error from true allele, related with homopolymers in repeats of STRs., (© 2024 Wiley‐VCH GmbH.)

Reducing the time required for DNA analysis in forensic genetics can yield significant benefits, both in determining genealogical relationships for legal proceedings and in criminal cases. Swift identification of individuals plays a pivotal role in solving crimes and apprehending perpetrators. Additionally, in situations like mass disasters, prompt victim identification holds utmost importance. The Rapid DNA technology, introduced in 2012 to expedite DNA analysis, has evolved to streamline the process into a single step. This advancement not only minimizes the risk of human error and contamination, but also boasts a remarkable time advantage, delivering results in as little as 90 min. In this study, DNA profiles of 30 families (consisting of mothers, fathers, and children) were analyzed using the RapidHITTM ID System. The system automatically calculated maternity-paternity probabilities to assess the suitability of Rapid DNA technology for kinship analysis. For validation, DNA profiles were also generated using the 3500 GA method. The study revealed that 9 out of 30 families exhibited discrepancies in DNA profiling, leading to inaccuracies in automatic kinship analysis. Consequently, while the method offers rapid and user-friendly advantages for forensic sciences, the software underlying the system requires re-evaluation. Issues such as maternal-paternal exclusion in kinship analyses, arising from challenges like un-called alleles, warrant further attention., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5-4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10 -9 . Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD 4 L did not., (© 2024. The Author(s).)

National forensic DNA databases are a valuable investigative tool, that have the potential to increase the efficacy of criminal investigations. Their unfettered expansion in recent years raises unsettling ethical issues that require close attention. DNA database expansion threatens the rights to privacy, non-discrimination, and equality, and can undermine public trust in government. This perspective piece relies on data from an international mapping study of Forensic DNA Databases to document the expansion of these databases, highlight the ethical issues they raise, and propose key recommendations for more responsible use of this infrastructure., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Short Tandem Repeat (STR) testing via capillary electrophoresis is undoubtedly the most popular forensic genetic testing method. However, its low multiplexing capabilities and limited performance with challenging samples are among the factors pushing scientists towards new technologies. Next-generation sequencing (NGS) methods overcome some of these limitations while also enabling the testing of Single-Nucleotide Polymorphisms (SNPs). Nonetheless, these methods are still under optimization, and their adoption into practice is limited. Among the available kits, Thermo Fisher Scientific (Waltham, MA, USA) produces three Precision ID Panels: GlobalFiler NGS STR, Identity, and Ancestry. A clear review of these kits, providing information useful for the promotion of their use, is, however, lacking. To close the gap, a literature review was performed to investigate the popularity, applications, and performance of these kits. Following the PRISMA guidelines, 89 publications produced since 2015 were identified. China was the most active country in the field, and the Identity Panel was the most researched. All kits appeared robust and useful for low-quality and low-quantity samples, while performance with mixtures varied. The need for more population data was highlighted, as well as further research surrounding variables affecting the quality of the sequencing results.

To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/μL), elevator buttons (11.9 pg/μL), cleaning supplies (7.17 pg/μL), and dispensers (16.4 pg/μL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10 -8 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations.

With the widespread use of the Y chromosome in genetics, a lot of commercially available Y chromosome kits were developed, validated, and applied to forensic science practice. The AGCU YNFS Y Kit is a new Y chromosome system containing forty-four preferred Y short tandem repeats (Y-STRs) and five common Y-InDels. In this study, the AGCU YNFS Y system was validated to verify its performance by following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM). A series of validation experiments included the following parameters: PCR-based studies, sensitivity studies, species specificity studies, stability studies, mixture studies, precision studies, stutter calculation, mutation and statistical analysis, population study, and case samples and degradation studies. The results suggested that appropriately changing PCR amplification conditions did not affect genotyping; the kit had good sensitivity for trace amounts of DNA (0.0625 ng), mixtures of multiple male individuals (minor: major = 1: 9), and three PCR inhibitors (more than 250 μM hematin, 250 ng/μL humic acid and 50 ng/μL tannic acid). The maximum standard deviation of allele size did not exceed 0.1552 reflecting the high accuracy of the system. By this, 87 DNA-confirmed pairs of father-son pairs were also analyzed for mutations. A total of 18 loci were mutated, with mutation rates ranging from 11.5×10-3 to 34.5×10-3 (95% CI 7.2×10-3-97.5×10-3, DYS627 and DYF404S1). In the population study, the haplotype diversity of 87 unrelated individuals was 0.9997, and discrimination capacity was 0.9885. Degradation studies have demonstrated that UV-C light exposure for up to 120 hours has no effect on male blood and semen-vaginal secretion mixtures. However, complete typing could no longer be obtained after 48 hours of UV exposure in single male saliva and in male saliva and female blood mixed samples. Collectively, the AGCU YNFS Y Kit is sensitive and accurate and can play its application value in forensic science practice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Height is known to be a classically heritable trait controlled by complex polygenic factors. Numerous height-associated genetic variants across the genome have been identified so far. It is also a representative of externally visible characteristics (EVC) for predicting appearance in forensic science. When biological evidence at a crime scene is deficient in identifying an individual, the examination of forensic DNA phenotyping using some genetic variants could be considered. In this study, we aimed to predict 'height', a representative forensic phenotype, by using a small number of genetic variants when short tandem repeat (STR) analysis is hard with insufficient biological samples. Our results not only replicated previous genetic signals but also indicated an upward trend in polygenic score (PGS) with increasing height in the validation and replication stages for both genders. These results demonstrate that the established SNP sets in this study could be used for height estimation in the Korean population. Specifically, since the PGS model constructed in this study targets only a small number of SNPs, it contributes to enabling forensic DNA phenotyping even at crime scenes with a minimal amount of biological evidence. To the best of our knowledge, this was the first study to evaluate a PGS model for height estimation in the Korean population using GWAS signals. Our study offers insight into the polygenic effect of height in East Asians, incorporating genetic variants from non-Asian populations., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Franz Kafka had beautiful eyes. So striking, that many of the famous author's friends and peers commented on them - but quite variously ('dark', 'brown', 'grey' & 'blue'). Eye colour as perceived by an observer is subjective, being influenced by physiological, environmental, and even sociocultural factors. In a policing context, this does not mean that trait information such as eye colour is not valuable (far from it), but that it must be managed carefully. The Australian Federal Police has recently implemented a forensic DNA phenotyping (FDP, aka. physical trait prediction or PTP) capability, utilising massively parallel sequencing DNA technology to predict an individual's eye colour, biogeographical ancestry and sex from a crime scene sample. This information alone is not itself 'intelligence', but can be used to generate intelligence through holistic analyses undertaken within a transdisciplinary, all-source forensic intelligence (FORINT) framework. FORINT outputs posit abductive propositions typically at the activity/offence level, to provide insight and influence decision making. However, the use of predicted traits requires that they are compared to something; all Australian police databases include fields for physical traits, but no uniform standard is applied across all agencies. Moreover, collection is inconsistent and no automated systems are in place to capture such data systematically. Consider the 'Kafka problem': his peers gave multiply divergent descriptions of his eyes. If a Biology unit had predicted the eye colour of an 'unidentified author' using DNA - how would Kafka be confidently nominated as the contributor? We posit three maxims for law enforcement: (1) To expand the operational utility of forensic science in line with police demands, forensic science should operationalise FDP (e.g. operationally to rank a list of persons of interest, focus lines of enquiry in serious & organised crime, or assist with human remains identification). (2) Such advanced biological techniques are best delivered through an all-source FORINT framework, to maximise opportunities and minimise risk. (3) One cannot pursue techno-scientific advancements in isolation; it is also necessary to influence the operational posture for their implementation. In this paper we explore these issues and provide recommendations relating to (a) police practices, (b) image capture systems, and (c) research opportunities. Phenotypic trait prediction has great potential and can be operationalised effectively through a rigorous FORINT framework. However, there is (continual) work to be done to enhance the operational capabilities that are complementary to - but necessary for - effective forensic science contribution to investigations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The genetic identification of skeletal remains from Chinese People's Volunteers (CPVs) of the Korean War has been challenging because of the degraded DNA samples and the lack of living close relatives. This study established a workflow for identifying CPVs by combining Y-chromosome short tandem repeats (Y-STRs), mitochondrial DNA (mtDNA) hypervariable regions I and II, autosomal STRs (aSTRs), and identity-informative SNPs (iiSNPs). A total of 20 skeletal remains of CPVs and 46 samples from their alleged relatives were collected. The success rate of DNA extraction from human remains was 100%. Based on Y-STRs, six remains shared the same male lineages with their alleged relatives. Meanwhile, mtDNA genotyping supports two remains sharing the same maternal lineages with their alleged relatives. Likelihood ratios (LRs) were further obtained from 27 aSTRs and 94 iiSNPs or 1936 iiSNPs to confirm their relationship. All joint pedigree LRs were >100. Finally, six remains were successfully identified. This pilot study for the systematic genetic identification of CPVs from the Korean War can be applied for the large-scale identification of CPVs in the future., (© 2024 Wiley‐VCH GmbH.)

DNA technology is the gold standard with respect to the identification of individuals from biological evidence. The technology offers the convenience of a universally similar approach and methodology for analysis across the globe. However, the technology has not realised its full potential in India due to the lack of a DNA database and lacunae in sample collection and preservation from the scene of crime and victims (especially those of sexual assault). Further, statistical interpretation of DNA results is non-existent in the majority of cases. Though the latest technologies and developments in the field of DNA analysis are being adopted and implemented,very little has been enacted practically to improve optimise sample collection and preservation. This article discusses current casework scenarios that highlight the pitfalls and ambiguous areas in the field of DNA analysis, especially with respect DNA databases, sampling, andstatistical approaches to genetic data analysis. Possible solutions and mitigation measures are suggested., 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 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

Microhaplotypes (MHs) were first recommended by Prof. Kidd for use in forensics because they can improve human identification, kinship analysis, mixture deconvolution, and ancestry prediction. Since their introduction, extensive research has demonstrated the advantages of MHs in forensic applications and provided useful data for different populations. Currently, two databases, ALFRED (ALlele FREquency Database) and MicroHapDB (MicroHaplotype DataBase), house the published MH information and population data. We previously constructed a single nucleotide polymorphism SNP-SNP MH database (D-SNPsDB) of MHs within 50 bp on the whole human genome for 26 populations integrating basic data such as physical genome positions, mapping of variant identifiers (rsIDs), allele frequencies, and basic variant information. Building upon the previous research, we further selected MHs containing at least two variants (SNPs and/or insertions/deletions [InDels]) within a short DNA fragment (≤ 50 bp) in 26 populations based on the 1000 Genomes Project dataset (Phase 3) to construct a more comprehensive database. Subsequently, we established a user-friendly website that allows users to search the MH database (MHBase) based on their research objectives and study population to find suitable loci and provides other functions such as querying reported loci, performing online calculations using the PHASE software, and calculating ancestral-related parameters. The loci in the database are classified as SNP-based MHs, which include only SNPs, and InDel-including MHs, which contain at least one InDel. Here, we provide a detailed overview of the MHBase and an analysis of shared loci at the global and continental levels, ancestral markers, the genetic distance within loci, and mapping with the genome annotation file. The website is an accessible and useful tool for researchers engaged in marker discovery, population studies, assay development, and panel design., 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 B.V. All rights reserved.)

The present study is aimed to address the challenge of wound age estimation in forensic science by identifying reliable genetic markers using low-cost and high-precision second-generation sequencing technology. A total of 54 Sprague-Dawley rats were randomly assigned to a control group or injury groups, with injury groups being further divided into time points (4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, and 32 h after injury, n = 6) to establish rat skeletal muscle contusion models. Gene expression data were obtained using second-generation sequencing technology, and differential gene expression analysis, weighted gene co-expression network analysis (WGCNA) and time-dependent expression trend analysis were performed. A total of six sets of biomarkers were obtained: differentially expressed genes at adjacent time points (127 genes), co-expressed genes most associated with wound age (213 genes), hub genes exhibiting time-dependent expression (264 genes), and sets of transcription factors (TF) corresponding to the above sets of genes (74, 87, and 99 genes, respectively). Then, random forest (RF), support vector machine (SVM) and multilayer perceptron (MLP), were constructed for wound age estimation from the above gene sets. The results estimated by transcription factors were all superior to the corresponding hub genes, with the transcription factor group of WGCNA performed the best, with average accuracy rates of 96% for three models' internal testing, and 91.7% for the highest external validation. This study demonstrates the advantages of the indicator screening system based on second-generation sequencing technology and transcription factor level for wound age estimation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Age prediction is an important aspect of forensic science that offers valuable insight into identification. In recent years, extensive studies have been conducted on age prediction based on DNA methylation, and numerous studies have demonstrated that DNA methylation is a reliable biomarker for age prediction. However, almost all studies on age prediction based on DNA methylation have focused on age-related CpG sites in autosomes, which are concentrated on single-source DNA samples. Mixed samples, especially male-female mixed samples, are common in forensic casework. The application of Y-STRs and Y-SNPs can provide clues for the genetic typing of male individuals in male-female mixtures, but they cannot provide the age information of male individuals. Studies on Y-chromosome DNA methylation can address this issue. In this study, we identified five age-related CpG sites on the Y chromosome (Y-CpGs) and developed a male-specific age prediction model using pyrosequencing combined with a support vector machine algorithm. The mean absolute deviation of the model was 5.50 years in the training set and 6.74 years in the testing set. When we used a male blood sample to predict age, the deviation between the predicted and chronological age was 1.18 years. Then, we mixed the genomic DNA of the male and a female at ratios of 1:1, 1:5, 1:10, and 1:50, the range of deviation between the predicted and chronological age of the male in the mixture was 1.16-1.74 years. In addition, there was no significant difference between the methylation values of bloodstains and blood in the same sample, which indicates that our model is also suitable for bloodstain samples. Overall, our results show that age prediction using DNA methylation of the Y chromosome has potential applications in forensic science and can be of great help in predicting the age of males in male-female mixtures. Furthermore, this work lays the foundation for future research on age-related applications of Y-CpGs., 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 B.V. All rights reserved.)

Poppies are beneficial plants with a variety of applications, including medicinal, edible, ornamental, and industrial purposes. Some Papaver species are forensically significant plants because they contain opium, a narcotic substance. Internationally trafficked species of illegal poppies are being identified by DNA barcoding employing multiple markers in response to their forensic value. However, effective markers for precise species identification of legal and illegal poppies are still under discussion, with research on illegal poppies focusing on Papaver somniferum L., and species identification studies of Papaver bracteatum and Papaver setigerum DC. still lacking. As a result, in order to evaluate the performance of genetic markers and classify their DNA sequences in the genus Papaver, this study developed the first machine learning-based two-layer model, in which the first layer classifies legal and illegal poppies from the given sequence and the second layer identifies species of illegal poppies using their sequences. We constructed the dataset and investigated biological features from four markers, internal transcribed spacer 1 (ITS1), internal transcribed spacer 2 (ITS2), transfer RNA Leucine (trnL), transfer RNA Leucine - transfer RNA Phenylalanine intergenic spacer (trnL-trnF intergenic spacer) and their combination, using four machine learning algorithms, K-nearest neighbor (KNN), Naïve Bayes (NB), extreme gradient boost (XGBoost) and Random Forest (RF). According to our findings, for Layer 1 to classify legal and illegal poppies, KNN-based models using combined ITS region achieved the greatest performance of accuracy 0.846 and 0.889 using training and test sets, respectively. Additionally, for Layer 2 to identify illegal poppy species, KNN-based models using combined ITS region achieved the best performance of 0.833 and 1.000 for using training and test sets, respectively. To validate the model, the combined ITS region, which includes ITS 1 and 2 sequences, from blind poppy samples were used as a case study, with the Layer 1 correctly classifying legal and illegal poppies with over 0.830 accuracy. Layer 2 correctly identified P. setigerum DC., however, only one of the three P. somniferum L. species was accurately identified. Nevertheless, our research shows that machine learning can be used to classify and identify legal and illegal poppy species using DNA barcodes which can then be used as an efficient and effective forensic tool for improved law enforcement and a safer society., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

The subject of inter- and intra-laboratory inconsistency was recently raised in a commentary by Itiel Dror. We re-visit an inter-laboratory trial, with which some of the authors of this current discussion were associated, to diagnose the causes of any differences in the likelihood ratios (LRs) assigned using probabilistic genotyping software. Some of the variation was due to different decisions that would be made on a case-by-case basis, some due to laboratory policy and would hence differ between laboratories, and the final and smallest part was the run-to-run difference caused by the Monte Carlo aspect of the software used. However, the net variation in LRs was considerable. We believe that most laboratories will self-diagnose the cause of their difference from the majority answer and in some, but not all instances will take corrective action. An inter-laboratory exercise consisting of raw data files for relatively straightforward mixtures, such as two mixtures of three or four persons, would allow laboratories to calibrate their procedures and findings., (© 2024 American Academy of Forensic Sciences.)

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution., Competing Interests: Declaration of Competing Interest The author(s) declare(s) that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Body fluids are one of the most encountered types of evidence in any crime and are commonly used for identifying a person's identity. In addition to these, they are also useful in ascertaining the nature of crime by determining the ty pe of fluid such as blood, semen, saliva, urine etc. Body fluids collected from crime scenes are mostly found in degraded, trace amounts and/or mixed with other fluids. However, the existing immunological and enzyme-based methods used for differentiating these fluids show limited specificity and sensitivity in such cases. To overcome these challenges, a new method utilizing microRNA expression of the body fluids has been proposed. This method is believed to be non-destructive as well as sensitive in nature and researches have shown promising results for highly degraded samples as well. This systematic review focuses on and explores the use and reliability of miRNAs in body fluid identification. It also summarizes the researches conducted on various aspects of miRNA in terms of body fluid examination in forensic investigations., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)