A novel 193-plex MPS panel integrating STRs and SNPs highlights the application value of forensic genetics in individual identification and paternity testing.

Massively parallel sequencing (MPS) has emerged as a promising technology for targeting multiple genetic loci simultaneously in forensic genetics. Here, a novel 193-plex panel was designed to target 28 A-STRs, 41 Y-STRs, 21 X-STRs, 3 sex-identified loci, and 100 A-SNPs by employing a single-end 400 bp sequencing strategy on the MGISEQ-2000™ platform. In the present study, a series of validations and sequencing of 1642 population samples were performed to evaluate the overall performance of the MPS-based panel and its practicality in forensic application according to the SWGDAM guidelines. In general, the 193-plex markers in our panel showed good performance in terms of species specificity, stability, and repeatability. Compared to commercial kits, this panel achieved 100% concordance for standard gDNA and 99.87% concordance for 14,560 population genotypes. Moreover, this panel detected 100% of the loci from 0.5 ng of DNA template and all unique alleles at a 1:4 DNA mixture ratio (0.2 ng minor contributor), and the applicability of the proposed approach for tracing and degrading DNA was further supported by case samples. In addition, several forensic parameters of STRs and SNPs were calculated in a population study. High CPE and CPD values greater than 0.9999999 were clearly demonstrated and these results could be useful references for the application of this panel in individual identification and paternity testing. Overall, this 193-plex MPS panel has been shown to be a reliable, repeatable, robust, inexpensive, and powerful tool sufficient for forensic practice.

Genetic polymorphism and variability in the Guangdong Hakka, Teochew, and Cantonese groups: A comprehensive analysis of 19 X-STRs.

BACKGROUND: X chromosomeshort tandem repeat (X-STR) loci are playing an increasingly important role inforensic work, identifying female traces in male contamination and explainingcomplex kinship analyses. METHODS: In this study, we analyzed thegenetic polymorphism of 19 X-STR loci in the Guangdong Hakka, Teochew and Cantonese groups, respectively, aswell as in the Guangdong Hakka, Teochew andCantonese pooled Han. The genetic diversity and forensic characteristics of the19 X-STRs and 7 linkage groups were investigated, respectively. RESULTS: The experiments showed that the genetic diversity (GD) and polymorphism information content (PIC) in the pooledGuangdong Han ranged from 0.5320 to 0.9234 and 0.4369 to 0.9171, respectively, and the cumulative power of discrimination for males (PDM), power of discrimination for females (PDF) and mean paternity exclusion chance (MEC) were higher than 0.9999999, indicating that the 19 X-STRs had high geneticpolymorphism and discriminatory power. Genetic differences among Chinese Hansubgroups and among different Chinese populations were investigated byphylogenetic reconstruction and principal component analysis (PCA), respectively. Genetic analyses based on neighbor-joining (NJ) tree and principal component analysis plot showed that Cantonese, Teochew and Hakka were closely genetically related, and different populations with closer linguistic components had more genetic affinity. CONCLUSIONS: This study adds to the forensic X-STR database and demonstrates the forensic efficiency of 19 X-STRs for the Hakka, Teochewand Cantonese populations in Guangdong, and the pooled Han of Hakka, Teochewand Cantonese people in Guangdong.

Diatoms pass through the gastrointestinal barrier and lead to false-positive: an animal experiment.

The diatom test has been used by forensic pathologist as standard for drowning, but the occurrence of false-positive results (presence of diatoms found in the tissues of subjects who died from causes other than drowning) draws criticism regarding the specificity of the test. Diatoms within food or water can be ingested through the gastrointestinal tract. However, the mechanisms of how the diatoms reach distant organs such as the lung, liver, and kidney have not been studied. In this article, we simulated the process of diatoms entering the gastrointestinal tract using gastric lavage on experimental rabbits. Diatoms are detected in lymph from a lymphatic vessel at the root of the mesentery, portal vein blood, aortic blood, lung, liver, and kidney samples in the gavage group. Of diatoms, 76.24% were the centric diatom, 99.86% of diatoms have a maximum size of less than 50 µm, and most of diatoms concentrate in the lung. Our study provided the evidence supporting the theory that the diatoms could pass through the gastrointestinal barrier and reach the rabbits' other internal organs. The diatoms could reach internal organs through the portal vein and lymphatic vessel at the root of the mesentery. This provides us new insight into our understanding of false-positive diatom test in forensic pathology.

Research Progress on Microbial Community Succession in the Postmortem Interval Estimation.

The postmortem interval (PMI) estimation is a key and difficult point in the practice of forensic medicine, and forensic scientists at home and abroad have been searching for objective, quantifiable and accurate methods of PMI estimation. With the development and combination of high-throughput sequencing technology and artificial intelligence technology, the establishment of PMI model based on the succession of the microbial community on corpses has become a research focus in the field of forensic medicine. This paper reviews the technical methods, research applications and influencing factors of microbial community in PMI estimation explored by using high-throughput sequencing technology, to provide a reference for the related research on the use of microbial community to estimate PMI.

Concordance analysis of diatom types and patterns in lung tissue and drowning medium in laboratory animal model.

Diatom test has been widely used in the diagnosis of drowning and inferring the drowning site. One of the issues is whether the concordance of the diatom types and patterns between the drowning victim's organs and media should be considered an essential requirement for the diagnosis of drowning. In this study, lung tissues from 20 rabbits and drowning media were studied by the Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy method, and four methods, type consistency, coefficient of similarity, squared-chord distance, and cluster analysis, were introduced to analyze the diatom types and patterns for evaluating the value of diatom consistency in drowning cases. The results showed that diatom types and patterns in lung tissues do not perfectly match the drowning medium, and they are sometimes concordant with the drowning medium sampled from other than drowning site. We should be cautious when using diatom detection to infer drowning sites.

Pathway of Diatoms Enter Experimental Rabbits through the Lymphatic System of the Digestive Tract.

OBJECTIVES: To study whether diatoms can enter the body through the lymphatic system of the digestive tract. METHODS: Twenty experimental rabbits were divided into the test group and the control group randomly, and intragastric administration was performed with 20 mL water sample from the Pearl River and 20 mL ultrapure water, respectively. After 30 min, lymph, lungs, livers and kidneys were extracted for the diatom test. The concentration, size and type of diatoms were recorded. RESULTS: The concentration of diatoms of the test group was higher than that of the control group (P<0.05). In the test group, Stephanodiscus, Coscinodiscus, Cyclotella, Melosira, Nitzschia, Synedra, Cymbella, and Navicula were detected; in the control group, Stephanodiscus, Coscinodiscus and Cyclotella were detected. The long diameter and the short diameter of diatoms of the test group were higher than those of the control group (P<0.05). In the test group, 1-2 diatoms were detected in 3 lung samples and 2 liver samples, which were Stephanodiscus or Cyclotella, and no diatoms were detected in the kidney samples; in the control group, 1-2 diatoms were detected in 2 lung samples and 3 liver samples, which were Stephanodiscus or Coscinodiscus, and no diatoms were detected in the kidney samples. CONCLUSIONS: Diatoms can enter the body through the lymphatic fluid, which is one of the reasons for the presence of diatoms in tissues and organs of non-drowning cadavers.

Effects of Digestive Temperature and Time on Diatom Test.

OBJECTIVES: To study the effects of temperature and time for diatoms digestion and find out suitable digestive temperature and time. METHODS: Eighty pieces of liver tissues were collected, each piece of tissue was 2 g, and 2 mL Pearl River water was added to each piece of tissue. The digestion temperature was set at 100 ℃, 120 ℃, 140 ℃, 160 ℃, 180 ℃ and the digestion time was set at 40, 50, 60, 70, 80 min. The liver tissue and water mixture were divided into 8 portions in each group. All the samples were tested by microwave digestive - vacuum filtration - automated scanning electron microscopy method. The quantity of diatom recovered and the quality of residue on the membrane were recorded. RESULTS: When the digestion time was set to 60 min, there were statistically significant differences in the number of diatoms recovered at different temperatures (P<0.05). The maximum number of diatoms recovered was (28 797.50±6 009.67) at 140 ℃, and the minimum residue was (0.60±0.28) mg at 180 ℃. When the digestion temperature was set at 140 ℃, there were statistically significant differences in the number of diatoms recovered at different digestion times (P<0.05). The number of diatoms recovered was the highest at 40 min, it was up to (20 650.88±1 950.29), and the residue quality of each group had no statistical significance among different digestion time groups(P>0.05). CONCLUSIONS: The effect of diatom digestion is related to temperature and time. When the digestion temperature was 140 ℃ and the digestion time was 40, 50 and 60 min, it is favorable for diatom test.

Application of Diatoms Quantitative Analysis in the Diagnosis of Drowning.

OBJECTIVES: To retrospectively analyze diatom test cases of corpses in water and discuss the value of quantitative analysis of diatoms in the diagnosis of drowning. METHODS: A total of 490 cases of water-related death were collected. They were divided into drowning group and postmortem immersion group according to the cause of death. Diatoms in lung, liver, kidney tissue and water sample were analyzed quantitatively by microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM) method. The ratios of content of diatoms in lung tissue and water sample (CL/CD) were calculated. RESULTS: The results of diatom test for three organs (lung, liver and kidney) were all positive in 400 cases (85.5%); the content of diatom in lung, liver, kidney tissues, and water samples of drowning group were (113 235.9±317 868.1), (26.7±75.6), (23.3±52.2) and (12 113.3±21 760.0) cells/10 g, respectively; the species of diatom were (7.5±2.8), (2.6±1.9), (2.9±2.1) and (8.9±3.0) types, respectively; the CL/CD of drowning group and postmortem immersion group were (100.6±830.7) and (0.3±0.4), respectively. CONCLUSIONS: Quantitative analysis of diatoms can provide supportive evidence for the diagnosis of drowning, and the parameter CL/CD can be introduced into the analysis to make a more accurate diagnosis of drowning.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning.

OBJECTIVES: To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning. METHODS: Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated. RESULTS: The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05). CONCLUSIONS: MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Development and application of a multiplex PCR system for drowning diagnosis.

In recent years, the DNA detection of drowning-related diatoms, cyanobacteria, and aeromonas has gradually attracted interest from forensic scientists. In this study, we described the validation and application of a novel multiplex PCR system. This system integrated 12 fluorescently labelled primers designed to amplify specific genes of diatoms, cyanobacteria, and aeromonas. The specificity studies demonstrated that this multiplex PCR system could detect nine species of diatom, seven species of cyanobacteria, and five species of aeromonas, all of which were drowning-related and widely distributed in various water circumstance of southern China. The sensitivity studies indicated that the limit concentration of template DNA was 0.0125 ng. Besides, this multiplex PCR system had good performance in sizing precision and stability, but it is not suitable for degraded DNA samples. The application into forensic casework showed that all the tissue samples from ten nondrowning cases showed negative results, and the positive rates of lung, liver, kidney, and water samples from 30 drowning bodies were 100, 86.7, 90, and 100%, respectively. Combined with results of diatom tests of MD-VF-Auto SEM method, this multiplex PCR system could help rule out nondrowning bodies and provide extra evidences to support drowning diagnosis, especially for those cases with few diatoms observed. It is expected that this multiplex PCR system has great potential for forensic drowning diagnosis.

The Y-STR landscape of coastal southeastern Han: Forensic characteristics, haplotype analyses, mutation rates, and population genetics.

The Y-STR landscape of Coastal Southeastern Han (CSEH) living in Chinese southeast areas (including Guangdong, Fujian, and Zhejiang provinces) is still unclear. We investigated 62 Y-STR markers in a reasonably large number of 1021 unrelated males and 1027 DNA-confirmed father-son pairs to broaden the genetic backgrounds of CSEH. In total, 85 null alleles, 121 off-ladder alleles, and 95 copy number variants were observed, and 1012 distinct haplotypes were determined with the overall HD and DC values of 0.999974 and 0.9912. We observed 369 mutations in 76 099 meiotic transfers, and the average estimated Y-STR mutation rate was 4.85 × 10-3 (95% CI, 4.4 × 10-3 -5.4 × 10-3 ). The Spearman correlation analyses indicated that GD values (R2 = 0.6548) and average allele sizes (R2 = 0.5989) have positive correlations with Y-STR mutation rates. Our RM Y-STR set including 8 candidate RM Y-STRs, of which DYS534, DYS630, and DYS713 are new candidates in CSEH, distinguished 18.52% of father-son pairs. This study also clarified the population structures of CSEH which isolated in population-mixed South China relatively. The strategy, SM Y-STRs for familial searching and RM Y-STRs for individual identification regionally, could be applicable based on enough knowledge of the Y-STR mutability of different populations.

Combined application of multiple autosomal and Y-chromosomal STR loci in solving a homicide case in 2009.

In 2009, a violent murder occurred. Two victims, a 47-year-old mother and her 21-year-old daughter, were murdered at home. After importing the 20 autosomal STR loci and 27 Y-STR loci into a database, no hit had been found. In 2019, a person with a prior criminal record was matched in the national forensic Y-STR database. When increasing the number of detected Y-STR loci to 60, all loci of the bloodstain donor at the crime and the suspect were still found to be identical. With the combined calculation of multiple autosomal STR and kinship index, we were able to identify the perpetrator as a previously unknown illegitimate child of a large family and solved the case.

The length and width of diatoms in drowning cases as the evidence of diatoms penetrating the alveoli-capillary barrier.

Forensic diatom test has been considered as a significant tool for diagnosis of drowning. Most of the studies in this field discussed the methodology of extracting, enriching and detecting diatoms from different tissues and drowning media. There are few studies on the basic principle of diatom test which was based on the theory developed by forensic scientists many years ago. This study was designed to analyze the length and width of diatoms in different organs and drowning medium samples of drowning cases. This study is designed to find evidence of diatoms penetrating the alveoli-capillary barrier. Samples from 100 drowning cases were analyzed using the methodology we developed: the Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy method (MD-VF-Auto SEM method). The results showed that the length and width of diatoms in the liver and kidney tissues are smaller than that of the lung tissues and water samples. Our studies also found that the pennate diatoms are easier to penetrate through the alveoli-capillary barrier, travel in the blood stream and finally deposit in the distant tissues including liver and kidney. These findings provided evidences to support the process of diatoms penetrating the alveoli-capillary barrier.

Linkage, recombination, and mutation rate analyses of 19 X-chromosomal STR loci in Chinese Southern Han pedigrees.

From Southern Han Chinese samples, we analyzed 19 X-STR markers for linkage, linkage disequilibrium (LD), and mutation rate. The data were collected from two- and three-generation Southern Han Chinese families. These data suggested that both linkage and linkage disequilibrium should be considered while calculating likelihood ratios with X-STR markers in relationship tests. The linkage disequilibrium of these 19 X-STR markers was calculated in our previous research study that was conducted on Southern Han Chinese population. In this study, the recombination fractions between pairs of markers and those obtained from the second-generation Rutgers combined linkage-physical map of the human genome were compared. The observed differences indicated that recombination was not homogeneous along the X chromosome. Therefore, we evaluated the effect on likelihood calculations by referring to haplotype frequencies obtained from allele distributions rather than haplotype counts of Southern Han Chinese population.

Analysis of false-positive results of diatom test in the diagnosis of drowning-would not be an impediment.

Diatom test has been a significant tool for the diagnosis of drowning. The reliability of the diatom test is still in strong dispute in the field of forensic science because of the false-positive results. This study was designed to quantitatively compare the numbers of the diatoms in false-positive cases and true drowning cases. Diatom samples from 64 victims were used in this study: 32 cases are confirmed drowning victims and other 32 cases died from non-drowned death. Samples were subject for the diatom test those were analyzed by the microwave digestion-vacuum filtration-automated scanning electron microscopy method (MD-VF-Auto SEM method) that we developed before. The results did show that there are false-positive diatoms detected in the liver and kidney tissues of non-drowned bodies: 6/20 in liver tissues and 7/20 in kidney tissues. However, the quantitative studies showed that there are statistical differences with the numbers of diatoms in the false-positive cases and in the true drowning cases. Diatom test of single organ is difficult for us to distinguish the sources of the diatoms detected. Therefore, comprehensive analysis of multiple organs would be more useful for the diagnosis of drowning.

Developmental validation of a 6-dye STR kit with 27 loci.

The EX27 System is a 6-dye (i.e., FAM, HEX, TAMAR, ROX, VIG, and SIZ-500) kit that combines the 24 autosomal STR loci (i.e., D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E, TPOX, TH01, D2S1338, CSF1PO, Penta D, D10S1248, D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, D22S1045, D2S441, SE33) with a Y-STR locus (DYS391), an Indel site (YIndel) and Amelogenin, which is designed to amplify DNA from extracts as well as direct amplification from substrates. The new system contains most of the widely used autosomal STR loci around the world and is sensitive to the male component in mixtures and is more accurate in gender determination owing to the DYS391 and YIndel.To demonstrate the suitability for personal identification and parentage testing, the system was validated for sensitivity of detection, species specificity, inhibitor tolerance, DNA mixtures, reproducibility study, and concordance studies based on the SWGDAM developmental validation guidelines. In view of the results, the EX27 System is a robust and reliable amplification kit which can be used for human identification testing.

Inference of drowning sites using bacterial composition and random forest algorithm.

Diagnosing the drowning site is a major challenge in forensic practice, particularly when corpses are recovered from flowing rivers. Recently, forensic experts have focused on aquatic microorganisms, including bacteria, which can enter the bloodstream during drowning and may proliferate in corpses. The emergence of 16S ribosomal RNA gene (16S rDNA) amplicon sequencing has provided a new method for analyzing bacterial composition and has facilitated the development of forensic microbiology. We propose that 16S rDNA amplicon sequencing could be a useful tool for inferring drowning sites. Our study found significant differences in bacterial composition in different regions of the Guangzhou section of the Pearl River, which led to differences in bacteria of drowned rabbit lungs at different drowning sites. Using the genus level of bacteria in the lung tissue of drowned rabbits, we constructed a random forest model that accurately predicted the drowning site in a test set with 100% accuracy. Furthermore, we discovered that bacterial species endemic to the water were not always present in the corresponding drowned lung tissue. Our findings demonstrate the potential of a random forest model based on bacterial genus and composition in drowned lung tissues for inferring drowning sites.

The Validation of a Single Multiplex Typing System With 45 Y-STR Markers for Familial Searching and Database Construction.

The Y-chromosomal short tandem repeat (Y-STR) is an effective forensic tool in familial searches and patrilineal relationship evaluation. However, currently available Y-STR panels often lack sufficient discriminatory power to resolve genetic relationships between distant relatives or within patrilocal populations. This study aims to establish a novel Y-STR amplification system for forensic casework analysis and database construction, which contains 44 slowly and moderately mutating and one rapidly mutating Y-STR. The validation of the assay was conducted following the recommendations of SWGDAM developmental validation guidelines. Different types of casework samples were tested and reliable profiles were obtained. Furthermore, we genotyped and analyzed 141 unrelated Han Chinese male samples. The results showed that this Y45 kit could improve the performance of identifying male individuals, higher haplotype diversity, and discrimination capacity when compared to the previous widely used Yfiler Plus kit. In general, the validation study demonstrated that the newly developed Y45 kit possesses high sensitivity, inhibitor tolerance, male specificity in a mixture, species specificity, and precision and is capable of forensic casework analysis and database construction.

An improved automated diatom detection method based on YOLOv5 framework and its preliminary study for taxonomy recognition in the forensic diatom test.

The diatom test is a forensic technique that can provide supportive evidence in the diagnosis of drowning but requires the laborious observation and counting of diatoms using a microscopy with too much effort, and therefore it is promising to introduce artificial intelligence (AI) to make the test process automatic. In this article, we propose an artificial intelligence solution based on the YOLOv5 framework for the automatic detection and recognition of the diatom genera. To evaluate the performance of this AI solution in different scenarios, we collected five lab-grown diatom genera and samples of some organic tissues from drowning cases to investigate the potential upper/lower limits of the capability in detecting the diatoms and recognizing their genera. Based on the study of the article, a recall score of 0.95 together with the corresponding precision score of 0.9 were achieved on the samples of the five lab-grown diatom genera via cross-validation, and the accuracy of the evaluation in the cases of kidney and liver is above 0.85 based on the precision and recall scores, which demonstrate the effectiveness of the AI solution to be used in drowning forensic routine.