Improving the Accuracy and Application of Nanopore DNA Sequencing
Noakes, Matthew Travis
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DNA contains the code of life, forming the molecular basis for all of life's diversity. The past several decades have witnessed remarkable progress in our ability to read and understand life's code through DNA sequencing. While fast and cheap DNA sequencing technologies are revolutionizing both science and healthcare, a new generation of technologies capable of single-molecule sequencing promise to further revolutionize the field of DNA sequencing by addressing many of limitations of the previous methods. Nanopore DNA sequencing is one such emerging single-molecule sequencing technology, capable of long reads and direct detection of epigenetically-relevant modified bases. The basic nanopore sequencing devices consists of two wells filled with a conductive electrolyte solution separated by an impermeable membrane containing a single nanometer-size hole, or nanopore. A voltage applied across the membrane drives an ionic current through the nanopore. DNA is negatively charged in solution and so will by drawn through the pore by the voltage, blocking some of the ionic current. As the different nucleotides along the DNA block the ionic current to different extents, the series of current fluctuations in the recorded time series can be used to decode the sequence of the DNA molecule moving through the pore. DNA motion through the pore is controlled using a DNA-processing motor enzyme, which steps the DNA through in discrete steps slow enough to allow resolution of the sequence-dependent fluctuations in the ionic current. Commercial nanopore sequencing devices have recently become available, making good on the decades-long promise of this technology. However, despite considerable early success and fanfare accompanying these first nanopore sequencers, technology development is not complete. Particularly, the single-read de novo sequencing accuracy must be improved for this technology to reach its full potential. In order to fully realize its promise, we must both improve the accuracy of nanopore sequencing and devise better methods of handling error-prone sequencing data. In this dissertation, I discuss my work in the Gundlach nanopore lab at the University of Washington towards the goals of improved nanopore sequencing accuracy and improved application of existing error-prone sequencing data. In chapter 1, I introduce the broad field of DNA sequencing. I cover the history of scientific interest in DNA and DNA sequencing and provide motivation for DNA sequencing as a worthwhile pursuit both for its scientific and medical merits. I also discuss previous and existing DNA sequencing technologies, as well as the limitations of these technologies that motivate the development of new methods such as nanopore sequencing. In chapter 2 I describe and introduce nanopore sequencing. I summarize the development of nanopore sequencing technology, how various challenges were overcome, and how currently available nanopore sequencing devices work, setting the stage for understanding the primary error modes limiting the sequencing accuracy of this technologies. In chapter 3, in I present my work on improving nanopore sequencing accuracy using a new method of DNA control for enzyme-actuated nanopore DNA sequencing. This new method, in which we use a time-varying voltage to control DNA motion through the pore in addition to a DNA-processing enzyme, is able to mitigate two of the primary error modes in nanopore sequencing and dramatically improve sequencing accuracy. I discuss the motivation behind this new method, outline how we were able to realize nanopore sequencing using this method, and demonstrate the improved sequencing accuracy it affords. In chapter 4, I shift the discussion over to my work on improving the application of nanopore sequencing data. Specifically, I introduce a method of aligning nanopore data that enables highly sensitive and specific sequence alignment and species identification even for low accuracy reads. I go over the motivation for this method, and present our findings of its improved performance over alternative methods. Finally, I conclude in chapter 5 where I discuss the implications of the demonstrated advances in the accuracy and application of nanopore sequencing, as well as look out towards further progress that can be made in both arenas.
- Physics