COMBO: An AmpliSeq-based trilogy for autosomal and uniparental (Y & mito) biogeographical ancestry and appearance testing

Forensic DNA phenotyping has become an increasingly important technology for narrowing down potential stain donors in forensic investigations. Over the past decade, the number of genetic markers required for reliable predictions has steadily increased, posing significant challenges for molecular genetic assay designs. These parallel designs often consume valuable DNA extracted from forensically relevant materials. To minimize DNA wastage, it is crucial to integrate as many markers as possible into a single analytical approach. Massively Parallel Sequencing (MPS)-based technologies have enabled the simultaneous analysis of up to several hundred genetic markers in one reaction. However, to date, marker panels from nuclear and mitochondrial DNA have not been integrated. This limitation has led to increased consumption of evidentiary DNA, as parallel analytical schemes are required to analyze both types of markers. This study presents the development, optimization and validation of the COMBO Panel, which combines autosomal and uniparental (Y-chromosomal and mitochondrial DNA) markers for ancestry and appearance testing. The COMBO panel contains 524 (VISAGE Enhanced Tool, ET) or 153 (VISAGE Basic Tool, BT) markers for the prediction of appearance and ancestry with 781 Y-chromosomal SNPs and 162 mtDNA amplicons covering the entire mitogenome. Library generation is based on AmpliSeq technology, which allows simultaneous analysis of all markers in a single sequencing run. The VISAGE ET panel was used in the optimization phase as well as in the initial trials of the COMBO assay and was later replaced with the VISAGE BT panel to streamline the test and adjust the primer concentrations accordingly. The COMBO panel was analyzed using Ion GeneStudio S5 systems for testing sensitivity and reproducibility, sex and species specificity, mock and real casework, degraded DNA and historical samples.