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DNA methylation patterns have the ability to reveal the activities of genes within a certain tissue at a particular time point. Tissue-specific DNA methylation patterns have been previously investigated for their applicability in the identification of forensically relevant body fluids, however there is still a lack in robust markers. While following a genome-wide scale investigation has a great potential to reveal useful tissue-specific changes, a gene-targeted approach can also lead to significant outcomes, especially in genomic locations not included in the genome-wide experiments. In this study, the potential of the candidate embryonal Fyn-associated substrate (EFS) gene for the positive identification of whole blood was investigated. For this purpose, the methylation profile of a selected genomic region containing a total of 10 CpG sites was analysed in 124 individuals via bisulfite pyrosequencing. Volunteers donated various forensically relevant tissues, including whole blood, saliva, seminal fluid, vaginal fluid and menstrual secretion. Whole blood showed the highest levels of DNA methylation (mean = 0.67), while semen samples were found to be very low methylated (mean = 0.06). The remaining tissues demonstrated partial mean methylation levels; more specifically, saliva − 0.43, vaginal fluid − 0.22 and menstrual blood − 0.22. One out of the 10 analysed CpG sites, CpG4, showed to be more robust, resulting in not only the highest methylation difference between blood and the rest of the tissues, but also the lowest inter-individual methylation difference. The proposed pyrosequencing assay was found to be accurate, linear and reproducible. Lastly, the method's applicability to forensic casework was assessed via the analysis of very old bloodstains stored up to 18 years, blood DNA samples stored long-term up to 9 years, mixed stains as well as other ‘forensic-like’ samples. In the majority of cases the expected methylation ratios were obtained indicating a stable D

The presence of specific body fluids at crime scenes could be linked with particular types of crime, therefore attributing a DNA profile to a specific tissue could increase the evidential significance of a match with a suspect. Current methodologies such as tissue-specific mRNA profiling are useful but drawbacks include low tissue specificity and applicability to degraded samples. In this study, the potential of 11 tissue-specific differentially methylated regions, initially identified following large-scale methylation analysis of whole blood, buccal cells and sperm, was explored in order to identify markers for blood, saliva and semen. Bisulphite pyrosequencing analysis supported previous findings, but tissue-specific differentially methylated regions for blood and buccal cells did not show enough specificity to be proposed as markers for blood and saliva, respectively. For some CpGs, a large inter-individual variation in methylation levels was also observed. Two of the semen markers (cg04382920 and cg11768416) were used for further validation on a large set of stains. These two semen-specific assays showed high sensitivity (as low as 50 pg) and stability. Future experiments will shed light on the usefulness of these markers in forensic casework.