This proposal concerns optimization of enzymes, pores, and computational methods for single molecule sequencing of genomic DNA fragments. It is based on a proven nanopore device implemented by our group at UCSC. This device is comprised of a sensor that touches and examines each nucleotide within a captured DNA strand as a processive enzyme motor advances the strand. Although the overall goal of nanopore sequencing is de novo reads on very long strands, here we will also focus on resequencing of DNA from organisms important in basic research (mouse, E. coli & Arabidopsis) and in healthcare (human). We are focusing on both de novo and resequencing for two reasons: 1) nanopore sequencing of biological DNA has not been documented publicly. Therefore, nanopore resequencing of reference standards is required for community acceptance, and, importantly, to reveal weaknesses in the technology that impact de novo sequencing accuracy; 2) nanopore resequencing in this application means reading genomic DNA directly and therefore will include epigenetic modifications. This would be an immediate, important contribution to the research community.
This proposal concerns a method for sequencing individual DNA molecules taken directly from the nucleus of a cell. It is based on a proven nanoscale device (a nanopore DNA strand sequencer) implemented by several research groups in the USA. This technique could have an immediate, important impact on medical research related to cancer and regenerative medicine.
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| Tyson, John R; O'Neil, Nigel J; Jain, Miten et al. (2018) MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome. Genome Res 28:266-274 |
| 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 |
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| 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 |
| Deamer, David; Akeson, Mark; Branton, Daniel (2016) Author response to John Kasianowicz and Sergey Bezrukov. Nat Biotechnol 34:482 |
| Jain, Miten; Fiddes, Ian T; Miga, Karen H et al. (2015) Improved data analysis for the MinION nanopore sequencer. Nat Methods 12:351-6 |
