Investigating the detection of two-person DNA mixtures using tri-allelic InDels

The analysis of complex DNA mixtures, such as unbalanced or degraded mixtures, remains a challenge in current forensic practice. InDel markers yield short amplicons and do not suffer from replication slippage in PCR amplification based on small-sized insertion or deletion fragments. In this study, the performance of tri-allelic InDels in analyzing two-person mixtures was evaluated. A total of 38 tri-allelic InDels with an inter-allelic length difference of ≥ 2 bp were characterized, and a multiplex amplification assay (FA-HID39plex) was developed with the sex identification marker (amelogenin). In comparison to biallelic InDels, these tri-allelic InDels generate higher likelihood ratios (LRs) for assessing whether a person of interest (POI) is included in a two-person DNA mixture. The FA-HID39plex assay enables the detection of the minor contributor in non-degraded two-person DNA mixtures at a 1:29 ratio. Although sensitivity decreases in degraded mixtures due to allele dropout, moderate support for the inclusion of the POI could still be obtained based on the LR method. Overall, tri-allelic InDels can serve as promising supplementary markers for the investigation of a POI in unbalanced or degraded two-person DNA mixtures through the calculation and evaluation of LRs.