William B. Eggleston*, Susan A. Greenspoon, and Cathryn A. Shannon | Virginia Department of Forensic Science Abstract: Processing evidence in sexual assault cases is time-consuming, which has led to high backlogs in many localities. The key challenge has been to separate DNA in sperm cells from DNA in epithelial cells prior to purification to simplify the mixture before DNA profiling. The differential extraction (DE) method, developed by Gill et al.1 and still routinely used in forensic laboratories, is based on the higher density of sperm cells and their resistance to cell lysis in the absence of a reducing agent such as dithiothreitol (DTT) relative to epithelial cells. Following lysis of epithelial but not sperm cells in buffers containing proteinase K and detergent, samples are centrifuged to pellet sperm cells (sperm fraction, or SF) leaving lysed epithelial cells in the supernatant (non-sperm fraction, or NSF). Removal of the NSF and repeated washing of the SF prior to DNA extraction is used to develop separate DNA profiles for the SF and NSF. Although effective, the process is a challenge to fully automate and the repeated wash steps are relatively slow, labor-intensive, can lead to loss of sperm, and provide more opportunities for cross contamination. Multiple procedures to improve the differential lysis/centrifugation method have been developed, including laser-, acoustic-, pH-, pressure-, affinity-, and enzyme-based methods. Critical aspects of new methods are to demonstrate that they work as well or better than methods in use in the forensic laboratory and have an ability to seamlessly integrate into existing workflows. Virginia Department of Forensic Science (VADFS) optimized and fully integrated an automated DNase I-based DE method into the current automated DNA processing pipeline using Promega’s DNA IQ™ DNA isolation system and Beckman Coulter’s Biomek® NXP automation workstation. DNase I is a nuclease that degrades DNA to oligonucleotides. Starting from reports by Garvin et al.2, 3 and Wong and Mihalovich4 and the VADFS’s current DE procedure, DNase I digestion of SFs was performed using a new method developed for the Biomek® NXP following manual epithelial cell lysis. The presenters report the results of testing multiple buffers, enzyme concentrations, incubation times and temperatures, and semen dilutions using vaginal and buccal mock sexual assault samples from multiple donors. The effects of commonly encountered vaginal contaminants and aging on results and tests to measure cross- contamination rates to validate the optimized protocol on the Biomek® NXP are also reported. This research found that multiple buffers and conditions worked as well as or better than the current VADFS standard DE protocol with respect to Y-DNA yields (measure of male DNA), ratios of autosomal-DNA to Y-DNA yields (A/Y ratios, which measure the relative quantity of female DNA), and the quality of sperm fraction DNA profiles. No evidence for increased rates of cross contamination was observed due to the automated DNase I treatment. The automated DNase I DE method is rapid and deposits the DNase I-treated SFs into the deep well sample plate, in which the SFs will be purified for DNA along with other casework samples using the existing automated extraction procedure. References 1. Gill, Peter, Alec Jeffreys, and David Werrett. “Forensic application of DNA ‘fingerprints’.” Nature 318 (1985): 577–579. https://doi.org/10.1038/318577a0. 2. Garvin, Alex M., Michael Bottinelli, Mauro Gola, Ario Conti, and Gianni Soldati. “DNA Preparation from Sexual Assault Cases by Selective Degradation of Contaminating DNA from the Victim.” Journal of Forensic Science 54, no. 6 (2009): 1297–1303. https://doi.org/10.1111/j.1556-4029.2009.01180.x. 3. Garvin, Alex M., Andrea Fischer, Jutta Schnee-Griese, Andrea Jelinksi, Michael Bottinelli, Gianni Soldati, Monica Tubio, Vincent Castella, Nathalie Monney, Naseem Malik, and Michelle Madrid. “Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach.” Investigative Genetics 3 (2012): 25. https://doi.org/10.1186/2041-2223-3-25. 4. Wong, Helena, and Jennifer Mihalovich. “Automation of the Differential Digestion Process of Sexual Assault Evidence.” Journal of Forensic Science 64 (2019): 539–550. https://doi.org/10.1111/1556-4029.13877.