The long-term objective of this project is rapid, highly accurate, and inexpensive sequencing of long (up to 150 kb) single DNA strands with a nanopore based DNA sequencing device. Meeting this long-term objective requires precise control of DNA movement past a nanopore sequence detector, and improvement of nanopore sequence detector resolution between DNA bases.
The specific aims of this proposal build upon progress made to date in these two areas by laboratories at the University of California, Santa Cruz (Akeson), University of Washington (Gundlach) and the University of Pennsylvania (Drndic). Individual laboratory expertise and knowledge will be integrated to accomplish four specific aims.
Specific aim one extends promising results at UCSC with DNA polymerase Phi29, a """"""""molecular step motor"""""""" able to precisely control DNA movement through a nanopore sequencer. This work will employ existing personnel and 12 years of success at UCSC with alpha hemolysin protein nanopores to extend understanding of Phi29 DNA polymerase function in a nanopore.
Specific aim two evaluates Phi29 DNA polymerase function with two nanopores selected for their potentially superior base resolution to the alpha hemolysin nanopore. The first is MspA, a protein nanopore, which will be evaluated with Phi29 DNA polymerase by U of Washington and UCSC teams. This collaboration takes advantage of expertise with use of Phi29 DNAP at UCSC and expertise with MspA at U of Washington. The second nanopore, a solid state ultrathin silicon nitride pore with fluorescence detection, will be evaluated with Phi29 DNA polymerase by U of Penn (makers of the solid-state nanopore) and UCSC teams. The third specific aim improves DNA base resolution through increased differences in current signals from individual bases. This will be achieved by increased salt concentrations in the nanopore combined with use of salt tolerant DNA Polymerases. DNA polymerases from salt tolerant organisms will be isolated by extremophile experts currently at UCSC.
Specific aim 4 will use all information gathered to generate proof of concept through sequencing of long (up to 48 KB) DNA strands using a nanopore sequencing device. Realization of this technology will provide the basis for a more complete understanding of individual genetic traits and predispositions in human and other populations.
This proposal develops nanopore based DNA sequencing for significant improvement in speed and fidelity of DNA sequencing over current technologies. The ultimate goal is sufficient speed and cost reduction to permit routine sequencing of individual genomes and ultimately provide the basis for a detailed understanding of individual genetic traits and predispositions. This understanding and technology are needed for personalized medicine in the 21st century.
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| Byrne, Ashley; Beaudin, Anna E; Olsen, Hugh E et al. (2017) Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. Nat Commun 8:16027 |
| Rand, Arthur C; Jain, Miten; Eizenga, Jordan M et al. (2017) Mapping DNA methylation with high-throughput nanopore sequencing. Nat Methods 14:411-413 |
| Jain, Miten; Olsen, Hugh E; Paten, Benedict et al. (2016) The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol 17:239 |
| Deamer, David; Akeson, Mark; Branton, Daniel (2016) Three decades of nanopore sequencing. Nat Biotechnol 34:518-24 |
| Carson, Spencer; Wanunu, Meni (2015) Challenges in DNA motion control and sequence readout using nanopore devices. Nanotechnology 26:074004 |
| Henley, Robert Y; Vazquez-Pagan, Ana G; Johnson, Michael et al. (2015) Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination. PLoS One 10:e0142155 |
| Jain, Miten; Fiddes, Ian T; Miga, Karen H et al. (2015) Improved data analysis for the MinION nanopore sequencer. Nat Methods 12:351-6 |
| Langecker, Martin; Ivankin, Andrey; Carson, Spencer et al. (2015) Nanopores suggest a negligible influence of CpG methylation on nucleosome packaging and stability. Nano Lett 15:783-90 |
| Schreiber, Jacob; Karplus, Kevin (2015) Analysis of nanopore data using hidden Markov models. Bioinformatics 31:1897-903 |
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