Important technical problems in nanopore sequencing have been overcome within the last five years, culminating in a practicable device with significant advantages, including the ability to sequence DNA strands 100,000 bases in length. Nevertheless, an individual nanopore would take more than a year to sequence a human genome. To sequence a genome in minutes, it is essential to sequence many thousands of DNAs in parallel. Our proposal addresses that issue by developing new methods to produce and monitor nanopore sequencing arrays. We will explore three general means to form arrays. First, we will examine arrays involving droplet interface bilayers (DIB). DIB arrays will be based on aqueous-aqueous, aqueous-hydrogel or hydrogel- hydrogel interfaces, and will be monitored by electrical or optical recording. In the latter case, each bilayer in the array will contain multple functional nanopores allowing a substantial increase in the rate of data acquisition. New lipid chemistry designed to stabilize the arrays will be a critical aspect of this approach. Second, bilayer-free systems will be fabricated by depositing protein pores in apertures in thin solid-stat films, notably silicon nitride. New chemistry for the derivatization of the surface oxide layer on silicon nitride will be developed to modify the apertures to accommodate the pores and to prevent current leaks between the pores and the aperture walls. Third, DNA nanostructures will be employed to build arrays. Nanopores suitable for sequencing applications will be constructed from DNA for use with either DIB or solid-state arrays. DNA nanopores or protein nanopores will also be attached to DNA tiles or scaffolds designed to maintain a pore-to-pore spacing suitable for optical detection. Finally, we will investigate nucleobase detection techniques compatible with the three classes of arrays. Advances in parallel electrical detection will be exploited. Optical approaches designed to greatly increase the number of pores that can be monitored will also be explored, including means to increase the field of view by using lens less wide-field detection. More speculatively super-resolution approaches will be investigated to determine whether the spacing between pores can be decreased into the sub-?m range. Our proposed studies build on strong preliminary data and our expertise in chemistry and chemical biology to develop new approaches to advance massively parallel nanopore sequencing. The sequencing technologies proposed here promise to deliver chips containing 104, and possibly 106 or more, functional pores. These chips will deliver not only a $1,000 genome, but an ultra-rapid genome in as little as 10 minutes.

Public Health Relevance

Single-molecule nanopore sequencing of DNA has significant advantages including the ability to sequence strands 100,000 bases in length. However, an individual nanopore would take more than a year to sequence a human genome. To sequence a genome in minutes, it is essential to sequence many thousands of DNAs in parallel, as addressed here with nanopore sequencing arrays.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
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Special Emphasis Panel (ZHG1-HGR-N (M1))
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Schloss, Jeffery
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Scripps Research Institute
La Jolla
United States
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