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.

BACKGROUND: Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator\'s conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures.
METHODS: The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios.
RESULTS: The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately.
CONCLUSIONS: A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

OBJECTIVE: To establish a rapid, accurate, and sensitive multiplex PCR detection method for the simultaneous identification of the six common edible meats (beef, lamp, chicken, pork, goose, duck), and to evaluate its application value in meat adulteration identification.
METHODS: Based on complete mitochondrial genomic sequences of six species in the GenBank database, DNA sequences (cattle：16S rRNA; sheep：COX-1; chickens：Cytb; pig：COX-1; goose：NADH2; duck：16S rRNA) with intra-species conservation and inter-species specificity were screened, and species-specific primers were designed to construct a multiplex PCR detection system that can simultaneously detect the meat of six common species. The species specificity, sensitivity and reproducibility of the system were studied, and the simulated mixture sample detection was performed.
RESULTS: This study successfully constructed a multiplex PCR detection system that can detect the meats of six common species simultaneously. The system was not effective in DNA amplification of non-target species. When the DNA template sizes were 0.062 5-2 ng/μL, the amplified products of all six species could be detected. The duck component was still detected when the mixing ratio of duck and beef was as low as 0.5%.
CONCLUSIONS: This study constructs and establishes a multiplex PCR detection system with strong specificity, high sensitivity, and good reproducibility. It can accurately identify the components of animal origin in common edible meats and provide a simple and practical method for identifying adulteration of common edible meats and meat products in China.
目的: 建立一种快速、准确、灵敏的多重PCR检测方法，用于同时鉴别6种常见食用肉类（牛、羊、鸡、猪、鹅、鸭），并评估其在肉类食品掺假鉴定中的应用价值。方法: 基于GenBank数据库中6个物种的线粒体全基因组序列，筛选具有种内保守性、种间特异性的DNA序列（牛16S rRNA、羊COX-1、鸡Cytb、猪COX-1、鹅NADH2、鸭16S rRNA），并设计物种特异性引物，以此构建一个可同时鉴别6种常见食用肉类的多重PCR检测体系。对该体系进行种属特异性、灵敏度和重复性研究，并进行模拟混合样品检测。结果: 成功构建了一个可同时鉴别6种常见食用肉类的多重PCR检测体系，该体系在非目标物种的DNA中均未有效扩增；在DNA模板量为0.062 5~2 ng/μL时，6个物种的扩增产物均能检见。在鸭肉和牛肉混合比例低至0.5%时，仍能检测到鸭肉成分。结论: 本研究构建了一个特异性强、灵敏度高、重复性好的多重PCR检测体系，可准确鉴别6种常见食用肉类食品中的动物源性成分，为我国常见食用肉类及肉制品的掺假鉴定提供了一种简单、实用的检测技术。.

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.

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.

Kinship analysis is a crucial aspect of forensic genetics. This study analyzed 1,222 publications on kinship analysis from 1960 to 2023 using bibliometric analysis techniques, investigating the annual publication and citation patterns, most productive countries, organizations, authors and journals, most cited documents and co-occurrence of keywords. The initial publication in this field occurred in 1960. Since 2007, there has been a significant increase in publications, with over 30 published annually except for 2010. China had the most publications (n = 213, 17.43%), followed by the United States (n = 175, 14.32%) and Germany (n = 89, 7.28%). The United States also had the highest citation count. Sichuan University in China has the largest number of published articles. The University of Leipzig and the University of Cologne in Germany exhibit the highest total citation count and average citation, respectively. Budowle B was the most prolific author and Kayser M was the most cited author. In terms of publications, Forensic Science International- Genetics, Forensic Science International, and International Journal of Legal Medicine were the most prolific journals. Among them, Forensic Science International-Genetics boasted the highest h-index, citation count, and average citation rate. The most frequently cited publication was \"Van Oven M, 2009, Hum Mutat\", with a total of 1,361 citations. The most frequent co-occurrence keyword included \"DNA\", \"Loci\", \"Paternity testing\", \"Population\", \"Markers\", and \"Identification\", with recent interest focusing on \"Kinship analysis\", \"SNP\" and \"Inference\". The current research is centered around microhaplotypes, forensic genetic genealogy, and massively parallel sequencing. The field advanced with new DNA analysis methods, tools, and genetic markers. Collaborative research among nations, organizations, and authors benefits idea exchange, problem-solving efficiency, and high-quality results.

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.

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.

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.

In paternity testing, when there are Mendelian errors in the alleles between the child and the parents, a slippage mutation, or silent allele may not fully explain the phenomenon. Sometimes, it is attributed to chromosomal abnormalities, such as uniparental disomy (UPD). Here, we present the investigation of two cases of suspected UPD in paternity testing based on short tandem repeat (STR) detection (capillary electrophoresis platform). Case 1 involves a trio, where all genotypes detected on chromosome 6 in the child are homozygous and found in the father. Case 2 is a duo (mother and child), where all genotypes on chromosome 3 in the child are homozygous and not always found in the mother. At the same time, Mendelian error alleles were also observed at specific loci in these two chromosomes. Furthermore, we used the MGIEasy Signature Identification Library Prep Kit for sequencing on the massively parallel sequencing platform, which included common autosomal, X and Y chromosomes, and mitochondrial genetic markers used in forensic practice. The results showed that the genotypes of shared STRs on the two platforms were consistent, and STRs and single nucleotide polymorphisms (SNPs) on these two chromosomes were homozygous. All other genetic markers followed the laws of inheritance. A comprehensive analysis supported the parent-child relationship between the child and the alleged parent, and the observed genetic anomalies can be attributed to UPD. UPD occurrences are rare, and ignoring its presence can lead to erroneous exclusions in paternity testing, particularly when multiple loci on a chromosome exhibit homozygosity.