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P306 – Mixture Accepted: Linking DNA Methylation and iSNP/STRs for Biological Material and Contributor Assignment
Assigning contributors to their corresponding biological material is a central challenge in mixture analysis. To address this, we verified the combined analysis of identity SNPs (iSNPs) located near eight tissue‑differentially methylated CpG positions. In addition, we demonstrated that STRs can be successfully examined together with adjacent DNA‑methylation sites. Six autosomal STRs (D1S1677, FGA, D5S2800, SE33, D8S1179 and D19S433) displayed differential DNA methylation across the different biological materials. Building on these results, we designed a 14‑marker, multi‑component multiplex assay and further characterized the loci in single‑source samples. The assay’s suitability for forensic casework was explored in mixture‑ and sensitivity experiments.
Semen, blood, menstrual blood, vaginal fluid, saliva, buccal mucosa and nasal secretion were collected from volunteers (n = 41–50 per material). DNA was extracted and quantified by qPCR. For mixture preparation, DNA extracts from different biological materials were combined in varying ratios. For sensitivity testing, the extracts were diluted to concentrations of 10 ng, 1 ng and 500 pg before bisulfite conversion. After bisulfite conversion, the 14 loci (six STRs and eight iSNPs) were analyzed with a multiplex‑amplicon‑based massive parallel sequencing (MPS) approach, also covering the surrounding methylation sites. Data were processed with FDSTools, using a previously developed library file adapted for bisulfite‑converted DNA to obtain allele‑linked DNA‑methylation levels.
Allele‑linked DNA‑methylation could be measured locus‑dependent down to 500 pg. The study showed that SNP genotypes, STR alleles, STR‑stutter artefacts and the cell‑type composition of the biological material affect DNA‑methylation levels. In the tested mixtures, contributors and their associated biological materials were assigned accurately, even when the mixtures were highly unbalanced. Finally, we propose a concept for interpreting mixtures and discuss the assay’s limitations.
Authors
- Laura Schmelzer (Institute of Forensic Medicine, University Medical Center Freiburg - Faculty of Medicine, University of Freiburg, Germany)
- Jana Naue (Institute of Forensic Medicine, University Medical Center Freiburg - Faculty of Medicine, University of Freiburg, Germany)
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