High Accuracy Nanopore Sequencing

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Principal Investigator/Program director (Last, first, middle): Gundlach, Jens, H. High Accuracy Nanopore Sequencing PROJECT SUMMARY The objective of this proposal is to enable nanopore sequencing technology to reach its fullest potential. With NHGRI funding, our group has played a pivotal role in developing nanopore sequencing. We paved the way to nanopore sequencing by first engineering the highly sensitive pore MspA to provide single-nucleotide resolution and then by controlling the DNA speed through MspA with an enzyme to give the first proof of concept of nanopore sequencing. In this application we outline techniques that will overcome the last hurdle to nanopore sequencing, low base calling accuracy. We request funding through this application to increase the single-passage base calling accuracy of nanopore sequencing by more than an order of magnitude to better than 99%. This goal is a transformative step forward for nanopore sequencing since it opens up ubiquitous applications of this technology in health care and beyond. Additionally, we will explore other improvements and variations that make nanopore sequencing less expensive and more useful. Our specific aims are: (1) implementing a hybrid-voltage-enzyme control of DNA that dithers DNA back and forth, thereby allowing identification and correction of enzyme error modes, while maximizing the information obtainable from the ion current readings, (2) improving the fundamental components of the nanopore sequencer by (i) improving the pore used for sequencing and (ii) teaming up with helicase experts to develop DNA-controlling enzymes specifically engineered to optimize nanopore sequencing quality, (3) developing techniques that permit high integration density of nanopores to increase sequencing throughput and to minimize DNA input quantity. Our team's success to date has enabled us to form partnerships with prominent collaborators and to gain support from many excellent labs in academia and industry, whose expertise assists us in bringing nanopore sequencing into health care. We will work with our partner labs to complete the aims outlined in this proposal. It is our goal to improve the quality of human life by delivering revolutionary sequencing technology that is accurate, fast and inexpensive. Project Summary