Developmental validation of the AGCU YNFS Y Kit: A new 6-dye multiplex system with 44 Y-STRs and 5 Y-InDels for forensic application.

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.

A multiplex microbial profiling system for the identification of the source of body fluid and skin samples.

Determining the source of body fluids is crucial in forensic investigations, as it provides valuable information about suspects and the nature of the crime. Microbial markers that trace the source of tissues and body fluids based on site specificity and temporal stability are often used effectively for this purpose. In this study, a multiplex system comprising seven microbial markers (Finegoldia magna, Corynebacterium tuberculostearicum, Cutibacterium acnes, Haemophilus parainfluenzae, Streptococcus oralis, Prevotella melaninogenica and Faecalibacterium prausnitzii) was developed to distinguish between skin, saliva, and feces samples. Based on these markers, the system produces electropherograms that are specific for each sample type. We collected 492 samples from six different skin sites (palm, antecubital crease, inguinal crease, cheek, upper back, and toe web space), the buccal mucosa, and stool were collected to further test the system. Beta diversity analysis revealed distinct clustering among the three sample groups. Additionally, skin microenvironment cluster analysis was used to identify skin sites accurately. This analysis classified skin samples into four distinct microenvironments: dry, moist, oily, and foot. Finally, we established a machine learning prediction model based on random forest regression to identify the skin microenvironment, achieving an overall prediction accuracy of 79 %. The multiplex system developed in this study accurately identifies the sources of body fluids, and the skin microenvironment. These findings offer new insights into the application of microbial markers in forensic science.

A high-throughput droplet digital PCR system aiming eight DNA methylation targets for age prediction.

The droplet digital Polymerase Chain Reaction (ddPCR) has garnered recognition for its distinctive attribute of absolute quantification. And it has found practical utility in age prediction through DNA methylation profiles. However, a prevalent limitation in current ddPCR methodologies is the restricted capacity to detect only two targets concurrently in most instruments, leading to high costs, sample wastage, and labor-intensive procedures. To address the limitations, a novel high-throughput ddPCR system allowing for the simultaneous detection of eight targets was developed. Through the implementation of a new 8-plex ddPCR assay, coupled with comprehensive linear regression analyses involving primers and probes ratios, diverse inputs of single CpG sites with distinct primers and probes, and varying plex assay configurations, stable DNA methylation values for four CpGs and stable measurement precisions for distinct multiplex systems were consistently observed. These findings pave the way for advancing the field of chemistry science by enabling more efficient and cost-effective methods. Furthermore, the comparative validation of ddPCR and SNaPshot demonstrated a remarkable concordance in results, and the system also displayed well in the field of various aspects, including species specificity, DNA input, and aged samples. In this study, the recommended input of bisulfite-converted DNA was determined to be 10-50 ng due to the double-positive droplets. Notably, the Pearson correlation coefficient squared values of four CpGs were 0.4878 (ASPA), 0.4832 (IGSF1), 0.6881 (COL1A1), and 0.6475 (MEIS1-AS3). And the testing set exhibited a mean absolute error of 4.5923 years, indicating the robustness and accuracy of the age-predictive model.

Explore variation of salivary bacteria across time and geolocations.

Saliva is an informative body fluid that can be found at various crime scenes, and the salivary bacterial community has been revealed it is a potential auxiliary target for forensic identification. However, the variation of salivary bacterial community composition across time and geolocation needs to be explored. The study was designed to be carried out during the winter vacation that was across about 50 days and eight geographic locations. The high throughput sequencing was performed with the V3-V4 region of the16S rRNA gene to explore salivary bacterial community composition. An overall slight fluctuation of the salivary bacteria was observed, which primarily occurred in the relative abundance of the salivary bacterial taxa. The results of principal coordinate analysis and hierarchical clustering showed samples were clustered by the individuals. All individuals could be correctly identified with the random forest model. In summation, although the relative abundance of salivary bacteria varied across the changes of time and geolocation, the individualized characteristic of salivary bacteria remained steady, which is beneficial for the salivary bacterial application in personal identification.

Characterization of loss of chromosome Y in peripheral blood cells in male Han Chinese patients with schizophrenia.

BACKGROUND: Schizophrenia (SCZ) has a global prevalence of 1% and increases the risk of mortality, reducing life expectancy. There is growing evidence that the risk of this disorder is higher in males than in females and it tends to develop in early adulthood. The Y chromosome is thought to be involved in biological processes other than sex determination and spermatogenesis. Studies have shown that loss of chromosome Y (LOY) in peripheral blood cells is associated with a variety of diseases (including cancer) and increased all-cause mortality. An analysis of the relationship between LOY and schizophrenia is warranted. METHODS: A total of 442 Chinese males (271 patients with schizophrenia vs. 171 controls) were included in this study. The copy numbers of the Y and X chromosomes were detected by positive droplets targeting the amelogenin gene (AMEL) on the Y chromosome and X chromosome (AMELY and AMELX, respectively), using droplet digital PCR (ddPCR). The LOY percentage was defined as the difference between the concentration of AMELX and the concentration of AMELY divided by the concentration of AMELX, denoted as (X - Y)/X. RESULTS: In the Han Chinese population, the LOY percentage was higher in the schizophrenia group than in the control group (p < 0.05), although there was no significant difference in the presence of LOY between the two groups. A strong correlation was found between the average of the disease duration and the average of the LOY percentage (R2 = 0.506, p = 0.032). The logistic regression analysis implied that the risk of LOY increases by 0.058 and 0.057 per year according to age at onset and duration of disease, respectively (ponset = 0.013, pduration = 0.017). CONCLUSIONS: In the Han Chinese population, the LOY percentage of the disease group was significantly different from that of the control group. The age of onset and duration of schizophrenia might be risk factors for LOY in peripheral blood cells. A larger sample size and expanded clinical information are needed for more in-depth and specific analyses.