Rachelle Turiello* | University of Virginia Abstract: The toolkit for forensic human identification (HID) has expanded beyond comparative methods for individualization to include investigative tools based upon nucleic acid (NA) analysis. The determination of chronological age from forensically relevant stains has garnered considerable attention, with over 300 research articles published and countless predictive models developed from the multiplexed analysis of few age-associated, epigenetic loci.1 Unfortunately, analysis of epigenetic “marks” requires both DNA extraction and chemical conversion of the DNA to conserve NA modifications for downstream analysis. The gold-standard techniques for both DNA extraction and chemical conversion by sodium bisulfite are time-consuming, labor-intensive, prone to analyst contamination with multiple tube transfers, and result in extensive DNA loss. In particular, sodium bisulfite conversion (BSC) is associated with extensive DNA damage, with even the most well-established methods resulting in more than 50% of template loss.2 Despite the development of more sensitive detection and analysis strategies downstream, including massively parallel sequencing (MPS), studies have demonstrated that the interrogation of epigenetic targets for forensic purposes requires increased sensitivity for reproducible and accurate predictions.3 To increase the analytical range associated with forensic epigenetic analysis, the presenter has developed a microfluidic system for the automated sample preparation that includes both DNA extraction and chemical conversion. The extraction step employs a thermophilic enzyme for lysis, producing an eluate in PCR-compatible buffer; this step omits the use of any solid phase materials to immobilize and wash the extract to conserve template.4 Released DNA proceeds through a miniaturized, dynamic solid phase BSC (dSP-BSC) process that has been previously optimized to conserve NA templates and maintain high conversion efficiency. All sequential unit operations are performed via a rotationally driven disc, roughly the size of a CD,5 in concert with a mechatronic system to drive fluid toward the periphery of the disc through centrifugal force, induce magnetic mixing,6 perform sacrificial valving,7 and complete targeted incubation steps through contact heating. The system was tested with epigenetic standards of known methylation status, human K-562 erythroleukemia cell lines, and human blood via multiple downstream strategies, including real-time polymerase chain reaction (RT-PCR), high-resolution melting (HRM), and pyrosequencing using age-associated targets FHL2 and ELOVL2 for proof of concept.8 Preliminary results confirm the extraction strategy is compatible with the dSP-BSC chemistry sans nucleic acid purification. Future work is focused on the complete automation of the protocol with the in-house engineered systems and optimization of the tool with forensically relevant samples. References: 1. Maulani, Chaerita, and Elza I. Auerkari. “Age Estimation Using DNA Methylation Technique in Forensics: A Systematic Review.” Egypt Journal of Forensic Science 10, no. 1 (2020):38. https://doi.org/10.1186/s41935-020-00214-2. 2. Leontiou, Chrysanthia A., Michael D. Hadjidaniel, Petros Mina, Pavlos Antoniou, Marios Ioannides, and Philippos C. Patsalis. “Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.” PLoS ONE 10, no. 8 (2015). https://doi.org/10.1371/journal.pone.0135058. 3. Aliferi, Anastasia, David Ballard, Mateo D. Gallidabino, Helen Thurtle, Leon Barron, and Denise Syndercombe Court. “DNA Methylation-Based Age Prediction Using Massively Parallel Sequencing Data and Multiple Machine Learning Models.” Forensic Science International: Genetics 37 (2018): 215–226. https://doi.org/10.1016/j.fsigen.2018.09.003. 4. Turiello, Rachel, Leah M. Dignan, Brayton Thompsonm Melinda Poulter, Jeff Hickey, Jeff Chapman, and James P. Landers. “Centrifugal Microfluidic Method for Enrichment and Enzymatic Extraction of Severe Acute Respiratory Syndrome Coronavirus 2 RNA.” Analytical Chemistry 94, no. 7 (2022): 3287–3296. https://doi.org/10.1021/acs.analchem.1c05215. 5. Thompson, Brandon, Yiwen Ouyang, Gabriela R. M. Duarte, Emanuel Carrilho, Shannon T. Krauss, and James P. Landers. “Inexpensive, Rapid Prototyping of Microfluidic Devices Using Overhead Transparencies and a Laser Print, Cut and Laminate Fabrication Method.” Nature Protocols 10 (2015): 875–886 https://doi.org/10.1038/nprot.2015.051. 6. Dignan, Leah M., M. Shane Woolf, Christopher J. Tomley, Aeren Q. Nauman, and James P. Landers. “Multiplexed Centrifugal Microfluidic System for Dynamic Solid-Phase Purification of Polynucleic Acids Direct from Buccal Swabs.” Analytical Chemistry 93, no. 19 (2021): 7300-7309. https://doi.org/10.1021/acs.analchem