Forensic science international. Genetics最新文献

Forensic scientists play a crucial role in assigning probabilities to evidence based on competing hypotheses, which is fundamental in legal contexts where propositions are presented usually by prosecution and defense. The likelihood ratio (LR) is a well-established metric for quantifying the statistical weight of the evidence, facilitating the comparison of probabilities under these hypotheses. Developing accurate LR models is inherently complex, as it relies on cumulative scientific knowledge. Ensuring transparency and rigor in these models is essential for building trust and fostering broader adoption. This is especially true in forensic genetics, where LRs are widely applied. Recently, the integration of Artificial Intelligence (AI), especially deep learning and machine learning, has introduced novel methods for predicting physical traits, ancestry, and age. However, unlike traditional approaches, many of these AI-driven methods function as "black boxes", raising concerns within the forensic community about potential biases, accountability, adversarial effects and other phenomena that could lead to erroneous outcomes. In this study, we use simulated scenarios as a proof-of-concept to illustrate two common applications of AI methods: (i) prediction of biogeographical ancestry and (ii) kinship inference. We critically examine cases where AI models can mislead forensic interpretation, which represents ethical and security challenges. We emphasize the need for rigorous evaluation and ethical oversight in the application of these methods.

It is challenging to distinguish monozygotic (MZ) twins using traditional autosomal STR genotyping due to their nearly identical genomes. As an important kind of small non-coding RNAs, microRNAs (miRNAs) are essential regulators of gene expression and considered as excellent biomarkers due to their resistance to degradation. Moreover, droplet digital PCR (ddPCR) has emerged as a powerful technique for detecting gene mutations and pathogenic microorganisms, owing to its sensitivity and reliability. We aimed to explore the differential expression of miRNAs between MZ twins using next-sequence platform and assess the reliability of differentially expressed miRNAs by ddPCR. MiRNA sequencing (miRNA-seq) revealed nine differentially expressed miRNAs shared across five pairs of twins, including hsa-miR-3620-3p, hsa-miR-6825-5p, hsa-miR-1273h-5p, hsa-miR-200a-5p, hsa-miR-3192-5p, hsa-miR-188-5p, hsa-miR-206, hsa-miR-4796-5p, and hsa-miR-6775-3p. Subsequently, the combination of real-time quantitative PCR (qPCR) and ddPCR confirmed the ability of five of these miRNAs (hsa-miR-1273h-5p, hsa-miR-3192-5p, hsa-miR-188-5p, hsa-miR-206, and hsa-miR-6775-3p) in distinguishing monozygotic twins. Furthermore, ddPCR demonstrated superior recognition accuracy compared to qPCR. Finally, we evaluated the degradation resistance of these five miRNAs under different environmental conditions. None of the five miRNAs showed a significant decrease in expression levels after being stored at room temperature for up to 180 days or undergoing 10 freeze-thaw cycles. In summary, our study revealed the potential application of miRNAs in differentiation of MZ twins and the powerful role of ddPCR in forensic medicine.

DNA and RNA markers are significant in forensic practices, such as individual and body fluid identification. However, forensic DNA and RNA markers were separately analyzed in most forensic experiments, which resulted in large amounts of sample consumption, complex procedures, and weak inter-evidence correlation. While several integrated methods based on capillary electrophoresis and next-generation sequencing technologies were reported, integrated procedures were mostly on nucleic acid co-extraction, co-electrophoresis, or co-sequencing, and the number and type of markers co-tested were limited. Four studies were conducted in this work to co-analyze DNA and RNA with a highly integrated next-generation sequencing-based workflow. First, six commercial kits for DNA and RNA co-extraction were compared, and two kits more suitable for total nucleic acid extraction were selected. Second, the feasibility of an experimental methodology for reverse transcription with total nucleic acid was investigated, and the results showed that reverse transcription reactions have no significant impact on DNA fragments used for forensic short tandem repeat and single nucleotide polymorphism genotyping. Third, a method for DNA and RNA library co-preparation was developed. Through the three studies, a highly integrated experimental workflow for targeted next-generation DNA and RNA sequencing was developed, including DNA and RNA co-extraction, reverse transcription with total nucleic acid, library co-preparation with genomic DNA and complementary DNA, co-sequencing, and data analysis. Fourth, to evaluate the workflow, we developed a multiplex panel consisting of 55 DNA markers (10 autosomal short tandem repeats and 45 autosomal single nucleotide polymorphisms) and eight messenger RNA markers (two peripheral blood-specific, four saliva-specific, and two housekeeping markers), and carried out validation experiments. High accuracy of DNA genotypes and RNA expression results were observed. Taken together, the novel integrated workflow could be used to sequence forensic DNA and RNA markers, which provides a promising method to comprehensively reveal DNA and RNA bioinformation with limited crime scene bio-materials.

Kinship determination is a valuable tool in forensic genetics, with applications including familial searching, disaster victim identification, and investigative genetic genealogy. Conventional typing of small numbers of autosomal short tandem repeats (STRs) confidently identifies only first-degree relatives. Massively parallel sequencing (MPS) can access more STRs and resolve alleles identical by length but differing in sequence (isoalleles), which may increase the power of kinship estimation, particularly when combined with additional sequenced single nucleotide polymorphism (SNP) loci, as in the ForenSeq DNA Signature Prep kit. MPS sequencing of ∼10,000 SNPs is available in the ForenSeq Kintelligence kit, promising detection of more distant kin, while SNP chips carrying hundreds of thousands of markers increase resolution still further. Here we evaluate these different resolutions in a set of pedigrees, and via simulations. As expected, the key factor influencing the precision of kinship estimation is the number of markers analysed and MPS-based analysis of STRs increases resolution, with the full set of ForenSeq DNA Signature Prep kit markers allowing detection of third-degree relatives. Since SNP chips include non-autosomal (X- and Y-chromosomal, and mitochondrial [mtDNA]) markers, we ask how these perform within the pedigrees, cross-referencing to Y-STR sequence data. We highlight the importance of understanding haplogroup resolutions in the increasingly complex Y and mtDNA phylogenies, to avoid false exclusions. Incorporation of X-SNPs allows tracing of X-chromosome segments within families. These different approaches can add value to kinship estimation, but some require simpler bioinformatic interfaces to make them more widely accessible in practice, and also access to appropriate allele frequency data to avoid problems associated with ancestry mis-specification.

The generation of forensic DNA profiles consisting of single nucleotide polymorphisms (SNPs) is now being facilitated by wider adoption of next-generation sequencing (NGS) methods in casework laboratories. At the same time, and in part because of this advance, there is an intense focus on the generation of SNP profiles from evidentiary specimens for so-called forensic or investigative genetic genealogy (FGG or IGG) applications. However, FGG methods are constrained by the algorithms for genealogical database searches, which were designed for use with single-source profiles, and the fact that many forensic samples are mixtures. To enable the use of two-person mixtures for FGG, we developed a workflow, MixDeR, for the deconvolution of mixed SNP profiles. MixDeR, a flexible and easy to use R package and Shiny app, processes ForenSeq Kintelligence® (QIAGEN, Inc.) SNP genotyping results and directs deconvolution of the profiles in EuroForMix (EFM). MixDeR then filters the EFM outputs to produce inferred single-source genotypes in reports formatted for use with GEDmatch® PRO. An optional MixDeR output includes metrics that assist with testing and validation of the workflow. As the Shiny app provides a graphical user interface and the software is designed to be run offline, MixDeR should be suitable for use by any laboratory developing FGG capabilities, no matter their bioinformatic resources or expertise.

Forensic science takes advantage of population variability in autosomal Short Tandem Repeat (STR) lengths to establish human identification. The most common method for DNA profiling by STR is based on PCR, where the highly polymorphic STR regions are amplified and analysed using Capillary Electrophoresis (CE) or Massively Parallel Sequencing (MPS). MPS determines not only the repeat length, but also the repeat structure and variations in the flanking regions, making this method superior in discriminatory power compared to CE. Reverse Complement PCR (RC-PCR) is a novel, more sophisticated PCR based MPS library preparation method combining indexing and PCR amplification in a single closed-tube reaction. In this document we describe the complete developmental validation of the IDseek® OmniSTR™ kit, an RC-PCR based MPS library preparation kit. The developed IDseek® OmniSTR™ kit contains 28 autosomal STR targets, one Y-chromosomal STR and the Amelogenin gene covering all relevant STR core loci from the USA, EU, UK and Interpol.

The ReAct (Recovery, Activity) project is an ENFSI (European Network of Forensic Science Institutes) supported initiative comprising a large consortium of laboratories. Here, the results from more than 23 laboratories are presented. The primary purpose was to design experiments simulating typical casework circumstances; collect data and to implement Bayesian networks to assess the value (i.e., likelihood ratio) of DNA results given activity level propositions. Two different experimental designs were used to simulate a robbery, where a screwdriver was used to force a door or window. Propositions and case information were chosen following laboratory feedback listing typical casework circumstances (included in the paper). In a direct transfer experiment, the defendant owned and used the screwdriver, but he did not force the door/window in question. An unknown person used the defendant's stolen screwdriver. In an indirect transfer experiment, the defendant neither owned, saw, nor used the screwdriver, nor did they force the door or window. For the second experiment, given the defence view, the defendant never held the screwdriver. We envisaged the situation where an object manipulated by the defendant (or the defendant himself/herself) would be touched by the unknown offender who would then force the window. It was found for the direct transfer experiment that unless a single contributor profile aligning with the known person's of interest profile was retrieved, the results did not allow to discriminate between propositions. On the other hand, for the indirect transfer experiment, both single and major contributor profiles that aligned with the person of interest (POI) supported the proposition that the person used the tool rather than an unknown person who had touched an object, when indeed the former was true. There was considerable variation in median recoveries of DNA between laboratories (between 200pg-5ng) for a given experiment if quantities are taken into account. These differences affect the likelihood ratios given activity level propositions. More than 2700 samples were analysed in the course of this study. Two different Bayesian Networks are made available via an open source application written in Shiny R: Shiny_React(). For comparison, all datasets were analysed using a qualitative method categorised into absent, single, major or other given contributors. The importance of standardising methods is emphasised, alongside the necessity of developing new approaches to assign the probability of laboratory-dependent DNA recovery. Freely accessible open databases play a crucial role in supporting these efforts.

Y-chromosomal short tandem repeats (Y-STRs) at rapidly mutating (RM) loci have been suggested as tools for differentiating paternally related males. RMplex is a recently developed system that incorporates 26 RM loci and four fast-mutating (FM) loci, targeting 44 male-specific loci. Here, we evaluated the RMplex by estimating Y-STR mutation rates and the overall differentiation rates for 542 Korean father-son pairs, as well as the genetic population values for 409 unrelated males. RMplex performed well, distinguishing 50.7 % of the father-son pairs by at least one mutation, a value 10 times higher than the previously reported differentiation rate achieved using the PowerPlex® Y23 System. Of the 369 mutations, 361 (97.8 %) were single-step mutations, with locus-specific mutation rates varying from 1.8 × 10^-3 to 1.1 × 10^-1 mutations per generation, and an average mutation rate of 2.3 × 10^-2. Gene diversity values ranged from 0.5696 for DYS442 to 0.9970 for DYF1000, and the haplotype discrimination capacity of unrelated males was 100 %. Among the loci studied, DYS712 exhibited the highest mutation rate in this study of the Korean population. Similarly, the mutation rate of this locus is reported to be substantially higher for the Japanese and Chinese populations than for European populations. These findings suggest that DYS712 mutations are relatively frequent in East Asian populations. Although we did not detect significant relationships among the Y-chromosome single nucleotide polymorphism-based haplogroups, allele length was strongly correlated with the mutation rate at DYS712, which is consistent with previous studies. Although the incorporation of multi-copy loci into RMplex contributed significantly to the high mutation rates detected and to its discrimination capacity, this requires careful interpretation, owing to the potential for duplications. Nonetheless, these findings provide evidence regarding the suitability of the RMplex for distinguishing paternally related males in the Korean population.