Investigating the epigenetic discrimination of identical twins using buccal swabs, saliva, and cigarette butts in the forensic setting

Monozygotic (MZ) twins are typically indistinguishable via forensic DNA profiling. Recently, we demonstrated that epigenetic differentiation of MZ twins is feasible; however, proportions of twin differentially methylated CpG sites (tDMSs) identified in reference-type blood DNA were not replicated in trace-type blood DNA. Here we investigated buccal swabs as typical forensic reference material, and saliva and cigarette butts as commonly encountered forensic trace materials. As an analog to a forensic case, we analyzed one MZ twin pair. Epigenome-wide microarray analysis in reference-type buccal DNA revealed 25 candidate tDMSs with >0.5 twin-to-twin differences. MethyLight quantitative PCR (qPCR) of 22 selected tDMSs in trace-type DNA revealed in saliva DNA that six tDMSs (27.3%) had >0.1 twin-to-twin differences, seven (31.8%) had smaller (<0.1) but robustly detected differences, whereas for nine (40.9%) the differences were in the opposite direction relative to the microarray data; for cigarette butt DNA, results were 50%, 22.7%, and 27.3%, respectively. The discrepancies between reference-type and trace-type DNA outcomes can be explained by cell composition differences, method-to-method variation, and other technical reasons including bisulfite conversion inefficiency. Our study highlights the importance of the DNA source and that careful characterization of biological and technical effects is needed before epigenetic MZ twin differentiation is applicable in forensic casework

Validated inference of smoking habits from blood with a finite DNA methylation marker set

Inferring a person’s smoking habit and history from blood is relevant for complementing or replacing self-reports in epidemiological and public health research, and for forensic applications. However, a finite DNA methylation marker set and a validated statistical model based on a large dataset are not yet available. Employing 14 epigenome-wide association studies for marker discovery, and using data from six population-based cohorts (N = 3764) for model building, we identified 13 CpGs most suitable for inferring smoking versus non-smoking status from blood with a cumulative Area Under the Curve (AUC) of 0.901. Internal fivefold cross-validation yielded an average AUC of 0.897 ± 0.137, while external model validation in an independent population-based cohort (

From forensic epigenetics to forensic epigenomics: Broadening DNA investigative intelligence

Human genetic variation is a major resource in forensics, but does not allow all forensically relevant questions to be answered. Some questions may instead be addressable via epigeno

Integrating the human microbiome in the forensic toolkit: Current bottlenecks and future solutions

Over the last few years, advances in massively parallel sequencing technologies (also referred to next generation sequencing) and bioinformatics analysis tools have boosted our knowledge on the human microbiome. Such insights have brought new perspectives and possibilities to apply human microbiome analysis in many areas, particularly in medicine. In the forensic field, the use of microbial DNA obtained from human materials is still in its infancy but has been suggested as a potential alternative in situations when other human (non-microbial) approaches present limitations. More specifically, DNA analysis of a wide variety of microorganisms that live in and on the human body offers promises to answer various forensically relevant questions, such as post-mortem interval estimation, individual identification, and tissue/body fluid identification, among others. However, human microbiome analysis currently faces significant challenges that need to be considered and overcome via future forensically oriented human microbiome research to provide the necessary solutions. In this perspective article, we discuss the most relevant biological, technical and data-related issues and propose future solutions that will pave the way towards the integration of human microbiome analysis in the forensic toolkit

Discovery of potential DNA methylation markers for forensic tissue identification using bisulphite pyrosequencing

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