Epigenetic Age Estimation by Detecting DNA Methylation Status in Buccal Swabs.

The analysis of DNA methylation (DNAm) levels at specific CpG sites represents one of the most promising molecular techniques for estimating an individual's age. To date, a considerable number of studies have reported the development of age prediction models on the basis of DNAm in body fluids, with only a few utilizing buccal swabs. The objective of this study was to identify age-dependent methylation CpG sites in three different genes (HOXC4, TRIM59, and ELOVL2) in buccal swab samples from the Chinese Han population. A total of 461 buccal swabs, with an age range of 0.4-80.8 years, were divided into a training set (n = 325) and a validation set (n = 136). Samples were analyzed by pyrosequencing in order to identify age-related genes with correlation coefficient. A random forest regression model was ultimately proposed, including eight CpGs in three genes, with a mean absolute error (MAE) of 2.119 years. The model performs independent validation set with an MAE of 4.391 years. Our findings illustrate that buccal swabs present a suitable alternative to biological traces for age prediction based on DNAm pattern using pyrosequencing and random forest regression, offering the additional advantage of being collected noninvasively.

A preliminary study characterizing temporal changes in soil bacterial communities after dismembered bones were buried.

Determining the burial time of skeletal remains is one of the most important issues of forensic medicine. We speculated that the microbiome of gravesoil may be a promising method to infer burial time by virtue of time-dependent. As we know, forensic scientists have established various models to predict the postmortem interval of a decedent based on the changes in body and soil microbiome communities. However, limited data are available on the burial time prediction for bones, especially dismembered bones. In this exploratory study, we initially conducted 16S rRNA amplicon high-throughput sequencing on the burial soil of 10 porcine femurs within a 120-day period and analyzed the changes in soil microbial communities. Compared with the control soil, a higher Shannon index in the microbial diversity of burial soil containing bones was observed. Correlation analysis identified 61 time-related bacterial families and the best subset selection method obtained best subset, containing Thermomonosporaceae, Clostridiaceae, 0319-A21, and Oxalobacteraceae, which were used to construct a simplified multiple linear regression model with a mean absolute error (MAE) of 56.69 accumulated degree day (ADD). An additional random forest model was established based on indicators for the minimum cross-validation error of Thermomonosporaceae, Clostridiaceae, 0319-A21, Oxalobacteraceae, and Syntrophobacteraceae, with an MAE of 55.65 ADD. The produced empirical data in this pilot study provided the evidence of feasibility that the microbial successional changes of burial soil will predict the burial time of dismembered bones and may also expand the current knowledge of the effects of bone burial on soil bacterial communities.

Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.