Research with human genetic data or human beings more generally requires valid informed consent. However, several exceptions exist to this universal principle, usually for situations where it is impossible or at least impractical to obtain this consent. Those exceptions are usually bound to requirements of necessity, shared benefit for the concerned community and minimization of harmful impact. Research without consent is normally subject to approval by an ethics review board. However, secondary use for research may also be based on statutory research privileges in the law. We identified several legal provisions in different countries, permitting the secondary use of DNA data that has been collected without consent for law enforcement purposes, which raises ethical questions. Most of the respective laws seem to be connected to privacy rules and permit the use of de-identified data only. However, whether individual DNA profiles in suspect and offender databases can actually be de-identified must be questioned. What appears to be less critical in terms of re-identification risk are the frequently mentioned "statistical indices", most likely referring to aggregated allele frequencies. However, reflections about data anonymity and a narrow purpose limitation to research aiming at the improvement of calculations of weight of evidence seem not to be the sole reasons for permitting research with entries from criminal offender databases. Distinctions between convicted offenders and other people in the database suggest an assumption of forfeiting some level of privacy protection upon conviction underlying some of these provisions. The use of entries on national DNA databases could therefore amount to problematic additional punishment.

IntroductionThe field of forensic DNA analysis has undergone rapid advancements in recent decades. The integration of massively parallel sequencing (MPS) has notably expanded the forensic toolkit, moving beyond identity matching to predicting phenotypic traits and biogeographical ancestry. This shift is of particular significance in cases where conventional DNA profiling fails to identify a single suspect. Supplementing forensic analyses with estimated biological age may be valuable but involves a complex and time-consuming DNA methylation analysis. This study explores and validates the performance of a comprehensive forensic third-generation sequencing assay utilizing Oxford Nanopore Technologies (ONT) in an adaptive and direct sequencing approach. We incorporated the most widely used forensic markers, i.e., STRs, SNPs, InDels, mitochondrial DNA (mtDNA), and two methylation-based clock classifiers, thereby combining forensic genetic and epigenetic analysis in one single workflow. Methods and resultsIn our investigation, DNA from six anonymous individuals was sequenced using the ONT standard adaptive direct sequencing approach, reaching a mean percentage of on-target reads ranging from 6.6 % to 7.7 % per sample. ONT data was compared to standard MPS data and Illumina EPIC DNA methylation profiles. Basecalling employed recommended ONT software packages. TREAT was used for ONT-based analysis of autosomal and Y-chromosome STRs, achieving 90–92 % correct calls depending on allelic read depth thresholds. InDel analyses for two lower-quality samples proved challenging due to inadequate read depth, while the remaining four samples significantly contributed to the observed percentage markers (60.9 %) and correct calls (97.8 %). SNP analysis achieved a 98 % call rate, with only two mismatches and two missed alleles. ONT-generated DNA methylation data demonstrated Pearson’s correlation coefficients with EPIC data ranging from 0.67 to 0.97 for Horvath’s clock. Additional age-associated markers exhibited Pearson’s correlation coefficients with chronological age between 0.14 (ELOVL2) and 0.96 (FHL2) at read depths of <30 and <20, respectively. Despite excluding mtDNA from our targeted sequencing approach, adaptive proof-reading fragments covered the complete mtDNA with an average read depth of 21–72, showing 100 % concordance with reference data. DiscussionOur exploratory study using ONT adaptive sequencing for conventional forensic and age associated DNA methylation markers showed high sequencing accuracy for a significant number of markers, showcasing ONT as a promising (epi)genetic forensic method. Future studies must address three critical aspects: determining clear quantity and quality measures and detection thresholds for accuracy, optimizing input DNA quantity for forensic casework expectations, and addressing ethical considerations associated with phenotype and ancestry analysis to prevent ethnic biases.