Analytical advantage of surface swabbing for male DNA recovery from mixed samples under female DNA interference

Male DNA detection from sexual assault evidence is often analytically challenging because biological samples collected from female victims typically contain abundant female epithelial cells and relatively few spermatozoa. Although differential extraction (DE) is routinely used to separate sperm and non-sperm cell fractions, the influence of the initial sampling strategy on downstream DNA recovery and short tandem repeat (STR) profile quality remains insufficiently evaluated. We compared surface swabbing and fabric cutting using 24 semen-positive underwear samples obtained from actual sexual assault cases and processed using a standard DE workflow. Analytical outcomes were assessed in terms of DNA recovery, female DNA background, and the interpretability of male STR profiles from the sperm fraction. Fabric cutting produced higher DNA amounts; however, much of the recovered DNA was derived from female epithelial cells, resulting in substantial background interference and mixed or poorly interpretable male STR profiles. In contrast, surface swabbing yielded lower DNA amounts but was associated with lower female DNA carryover and improved male STR interpretability. The median male: female DNA ratio was approximately 6.8-fold higher with surface swabbing, which yielded interpretable male DNA profiles in 20 of 24 cases compared with 13 of 24 cases for fabric cutting. Under the conditions examined in this study, surface swabbing appears to be a preferable first-line sampling approach for semen-positive underwear evidence, while fabric cutting may be used as a complementary option in specific cases. These findings highlight the importance of optimizing the initial sampling step in DE-based forensic DNA workflows.