Comparison of automated and manual DNA differential extraction in mock vaginal sexual assault swabs

Differential extraction is a core forensic DNA technique separating sperm cells from epithelial cells in sexual assault evidence, facilitating recovery of male DNA profiles from mixed biological samples. Effective physical separation reduces the occurrence of mixed DNA profiles, simplifies interpretation, and conserves analyst time. This process is especially critical for samples with low sperm cell counts, where successful recovery of a probative male profile may be limited. This study evaluated and compared manual and robotic differential extraction methods using 30 mock forensic vaginal swabs with microscopic sperm ratings ranging from 4+ to <1+, representative of sexual assault samples encountered in forensic casework. Swabs were divided into equal portions and extracted in duplicate using both methods by two analysts for reproducibility. Manual and robotic differential extractions were performed using the Qiagen QIAamp® DNA Investigator Kit and standardized "Isolation of Total DNA from Sexual Assault Specimens" protocol for reagent and procedural consistency. Robotic extractions were conducted using the Qiagen QIAcube®. Performance metrics included DNA yield, DNA mixture incidence in epithelial and sperm fractions, and STR profile completeness. Robotic extraction demonstrated procedural consistency and reduced hands-on processing time. However, manual differential extraction produced superior separation performance, with a lower incidence of DNA mixtures in sperm fractions. Samples processed using robotic protocols were significantly more likely to exhibit epithelial carryover into sperm fractions, complicating interpretation and resulting in inconclusive results. In contrast, manual extraction yielded cleaner sperm fractions and improved recovery of single-source male DNA profiles, highlighting the trade-offs between automation and manual separation.