Correction: Methylation Markers for the Identification of Body Fluids and Tissues from Forensic Trace Evidence.

[This corrects the article DOI: 10.1371/journal.pone.0147973.].

Methylation Markers for the Identification of Body Fluids and Tissues from Forensic Trace Evidence.

The identification of body fluids is an essential tool for clarifying the course of events at a criminal site. The analytical problem is the fact that the biological material has been very often exposed to detrimental exogenous influences. Thereby, the molecular substrates used for the identification of the traces may become degraded. So far, most protocols utilize cell specific proteins or RNAs. Instead of measuring these more sensitive compounds this paper describes the application of the differential DNA-methylation. As a result of two genome wide screenings with the Illumina HumanMethylation BeadChips 27 and 450k we identified 150 candidate loci revealing differential methylation with regard to the body fluids venous blood, menstrual blood, vaginal fluid, saliva and sperm. Among them we selected 9 loci as the most promising markers. For the final determination of the methylation degree we applied the SNuPE-method. Because the degree of methylation might be modified by various endogenous and exogenous factors, we tested each marker with approximately 100 samples of each target fluid in a validation study. The stability of the detection procedure is proved in various simulated forensic surroundings according to standardized conditions. We studied the potential influence of 12 relatively common tumors on the methylation of the 9 markers. For this purpose the target fluids of 34 patients have been analysed. Only the cervix carcinoma might have an remarkable effect because impairing the signal of both vaginal markers. Using the Illumina MiSeq device we tested the potential influence of cis acting sequence variants on the methylation degree of the 9 markers in the specific body fluid DNA of 50 individuals. For 4 marker loci we observed such an influence either by sole SNPs or haplotypes. The identification of each target fluid is possible in arbitrary mixtures with the remaining four body fluids. The sensitivity of the individual body fluid tests is in the same range as for the forensic STR-analysis. It is the first forensic body fluid protocol which considers the exogenic and endogenic parameters potentially interfering with the true results.

MtDNA typing of single-sperm cells isolated by micromanipulation.

Some sexual assault crimes constitute a problem for the legal institutions confronted with the DNA analysis of such cases. Often, sperm cells are found in the victim's vaginal tract during medical examination but their successful genotyping is compromised by the huge excess of the victim's epithelial cells as well as by the degradation of genomic DNA present in sperm cells as a consequence of female immune response. Mitochondrial DNA present in the mid-piece of sperm cells might be useful in some specific cases in order to differentiate the donors of a semen sample. The high number of copies per cell and its circular nature that may confer some protection from the action of exonucleases make it more suitable for cases where few cells are available and/or the DNA is degraded. We have developed a novel strategy for typing mtDNA from single-sperm cells. Specific amplification of male mitochondrial DNA is ensured by use of sequence specific primers designed on the basis of mitochondrial single nucleotide polymorphisms existent throughout the control region. The strategy was applied to single-sperm cells isolated by micromanipulation from slides smeared with vaginal swabs taken immediately after sexual intercourse of voluntary couples. After sequencing the PCR products, it was possible to obtain a match between the DNA sequence from the buccal swab and the DNA sequence of the single sperm-cell, for each voluntary man. With this new strategy, the problem of contamination with DNA from the victim observed when using universal primers was completely overtaken. This method will probably allow the resolution of multiple-rapist crimes, where the collected sperm cells can be separately typed.