A step forward in comprehensive nanopore-based forensic profiling of STRs, InDel, SNP and mtDNA for identity and appearance prediction.

The targeted analysis of diverse DNA markers, including single nucleotide polymorphisms (SNPs), insertion deletion polymorphisms (InDels) and DNA methylation levels, is central to forensic DNA phenotyping, enabling inference of BGA, appearance traits, and age. Such information is relevant when DNA comparison after conventional DNA profiling fails to identify a person of interest. Recent technological developments allow both genetic and epigenetic markers to be analyzed in long-reads within a single platform such as Oxford Nanopore Technologies (ONT). This approach offers an advantage over traditional capillary electrophoresis and targeted, short-read massively parallel sequencing (MPS) methods by allowing genetic, and epigenetic marker detection in one assay. These (epi)genetic markers for DNA phenotyping have previously been evaluated using the ONT MinION platform, which may improve upon using the PromethION device with a larger pore count which we evaluated in this study. We included not only appearance and ancestry prediction, but also lineage and individual identification. Both whole-genome sequencing (WGS) and adaptive sampling were applied in which STRs, SNPs, InDels, and whole mtDNA were analyzed under both standard (600 ng) and lower-input (<100 ng) conditions. Subsequently, FDSTools, a bioinformatic tool designed for MPS-based forensic marker analysis, and Clair3, a tool for identifying genetic variants in long-read data, were assessed for their suitability in processing nanopore sequencing data. STR allele calling using FDSTools with an allelic balance threshold of 0.25 achieved the highest performance, with F1-scores corresponding to accuracies of 89.1-99.2% across the experimental setups. For InDels, FDSTools also performed best, yielding an F1-score of 98.1%. SNP analysis of 1231 autosomal, X-, and Y-chromosomal markers achieved F1-scores of 98.1-99.8% after applying additional quality thresholds. MtDNA analysis demonstrated F1-scores of 85.9-88.6% for whole mtDNA, and 100% for the control region. Overall, adaptive sampling with standard DNA input produced the highest precision, recall, and F1-scores across all variant types, followed by WGS and lower-input adaptive sampling. While slight variation in appearance probabilities and ancestry proportions were observed due to low level sequencing errors, no differences in appearance and ancestry conclusions between ONT and reference data were seen. Our findings demonstrate the potential of the PromethION platform for integrated forensic DNA analysis by enabling simultaneous analysis of numerous genetic markers of different types for different forensic purposes.