In recent years rapid improvements in DNA sequencing technology have positively and dramatically impacted biomedical research in the US and around the world. This progress is on-going, as the capabilities of DNA sequencing instruments continue to increase, while their cost continues to decline. Moreover, DNA sequencing technology is now on the verge of directly impacting health care as it becomes an integral part of clinical practice. Sequencing of DNA, however, first requires its isolation from is normal biological context, as well as its adaptation to a form compatible with sequencing technology. These activities are referred to as sample and library preparation, respectively. Recently it has become clear that the costs and logistical burdens associated with sample and library preparation are now, and will increasingly in the future, negatively impact dissemination of DNA sequencing technology. This is because, unlike DNA sequencing itself, which is automated within increasingly efficient DNA sequencing instruments, the costs and user burdens associated with sample and library preparation have not declined significantly. The overall aim of this Phase II SBIR application is to eliminate this critical bottleneck for both research and clinical DNA sequencing applications. GenapSys has developed innovative Digital Pneumatic Microfluidic (DPM) technology to address directly the cost and user-burden deficiencies of existing sample and library preparation methods. DPM chips and instruments promise to transform expensive and logistically burdensome sample and library preparation tasks to a convenient plug-and-play format that will reduce costs in existing applications and remove barriers to deployment of new ones. Unlike previous microfluidic technologies, DPM chips will be fabricated from low-cost materials, and with low-cost mass-production methods. They will be manufactured pre-loaded with virtually all the chemicals and reagents necessary for their function, and will reduce costs further by requiring only small quantities of each of these. n addition DPM instruments will be relatively inexpensive and compact, requiring only a small space in the laboratory or clinic. The specific aims of this Phase II SBIR proposal are designed to de-risk commercialization of DPM technology by optimizing our existing methods and designs, and by providing additional proof-of-concept for certain important features. This work involves optimization of our designs and manufacturing methods for microfluidic pumps and valves, improved high-temperature performance, and demonstration of methods to package and store chemicals and reagents on-chip. The proposed work culminates in demonstration of reliable and high quality library preparation by DPM chips and prototype instruments.