More than just blood, saliva, or sperm—setup of a workflow for body fluid identification by DNA methylation analysis

The determination of cellular origin of DNA is a useful method in forensic genetics and complements identification of the DNA donor by STR analysis, since it could provide helpful information for the reconstruction of crime scenes and verify or disprove the descriptions of involved people. There already exist several rapid/pre-tests for several secretions (blood, sperm secretion, saliva, and urine), RNA-based expression analyses (blood, menstrual blood, saliva, vaginal secretion, nasal secretion, and sperm secretion), or specific CpG methylation analyses (nasal blood, blood, saliva, vaginal secretion, nasal secretion, and sperm secretion) for determining the cell type. To identify and to discriminate seven different body fluids and mixtures thereof in a simple workflow from each other, assays based on specific methylation patterns at several CpGs combined with pre-/rapid tests were set up in this study. For each of the seven secretions listed above, we selected the CpG marker achieving the highest possible discrimination (out of 30 markers tested). Validation studies confirmed a definite identification for saliva, vaginal secretion, and semen secretion in 100% of samples as well as discrimination from all other secretions. Moreover, the unambiguously correctly determined proportion of nasal samples, blood and menstrual blood varied between 61% (nasal blood) and 85% (nasal secretion). In summary, our workflow proved to be an easy and useful tool in forensic analysis for the identification and discrimination of seven different body fluids often found at a crime scene. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00414-023-03069-z.