We are requesting an administrative supplement to purchase equipment for R35 award 5R35GM133483 “Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells” (project period: 09/17/2019 - 07/31/2024). We are requesting to purchase a Bio-Rad Automated Droplet Generator AutoDG Instrument (Bio- Rad item #1864101) for use with a QX200 Droplet Digital PCR (ddPCR) System and an associated Bio-Rad PX1 PCR Plate Sealer (Bio-Rad item #1814000) in order to significantly enhance the throughput and reproducibility of sample processing for the QX200 ddPCR system currently housed at the Joint School of Nanoscience and Nanoengineering (JSNN) of North Carolina A&T State University and UNC Greensboro (UNCG), where the PI is faculty (at UNCG and JSNN). ddPCR is an advanced PCR technique where individual nucleic acids from a sample are encapsulated in small oil-water emulsions prior to amplification by PCR, so that after the PCR cycles the emulsions that originally contained individual nucleic acids can be counted using fluorescent detection. ddPCR is at this point an established nucleic acid quantification technology that leads in precision and accuracy. Compared to traditional quantitative PCR (qPCR) methods such as the standard Applied Biosystems 7500 Real-Time PCR instrument (also at JSNN), ddPCR allows for absolute quantification of specific nucleic acids in a sample without the need for or variability of standard reference curves; provides increased sensitivity for the detection of rare mutants by over an order of magnitude (from >5% prevalence using qPCR to <0.1% prevalence); and exhibits less sensitivity to PCR inhibitors. These attributes will be crucial for emerging applications in projects related to the R35 grant. ddPPR and these requested equipment are expected to significantly help to advance and enhance several parallel lines of research associated with the R35 award in my laboratory. The reason for this is that the limitation of our ddPCR system is in its sample preparation—while the QX200 can perform nucleic acid quantification of 96 samples at a time, the standard equipment performs droplet preparation for only 8 samples at a time and this must be performed manually. The AutoDG system automates the simultaneous preparation of 96 samples, allowing us to maximize usage of this instrument and associated consumables. This automated processing will be necessary for the R35 research to significantly improve sample preparation throughput for the QX200 ddPCR system by orders of magnitude while reducing intra- and inter-user variability in preparation, all of which will be necessary to the completion of the research in a timely manner, to the highest quality standards, and at the sensitivity necessary to resolve key differences between experimental conditions. The PX1 PCR plate sealer will be necessary to maintain biosafety level- appropriate containment of Biosafety level 2 (BSL2) samples prior to and after droplet preparation.