Three decades of nanopore sequencing

Abstract

A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobase-by-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.

Figure 1

Nanopore sequencing. (a) Two ionic solution-filled chambers are separated by a voltage-biased membrane. A single-stranded polynucleotide (black) is electrophoretically driven through an MspA nanopore (green) that provides the only path through which ions or polynucleotides can move from the cis to the trans chamber. Translocation of the polynucleotide through the nanopore is controlled by an enzyme (red). (b) Portion of a record showing the ionic current through a nanopore measured by a sensitive ammeter. In nanopore strand-sequencing, the stepping rate is usually 30 bases per second, but this experiment was carried out using exceptionally low concentrations of ATP to slow the helicase activity, thereby increasing the duration of each level to illustrate the resolution that can be achieved.

Figure 2

Milestones in nanopore DNA sequencing.

Figure 3

D.D.’s notebook. Two pages of a notebook in which D.D. sketched the original nanopore sequencing concept are shown (dated June 25, 1989). The sketch shows three bases of a DNA strand being drawn through a nanopore by an applied voltage. The voltage also produces an ionic current, and the expected base-specific electrical readout is shown below with each base affecting the current in such a way that the sequence of bases can be determined.

Figure 4

Sensing regions in MspA and α-hemolysin. The sensing region in MspA is much shorter than in α-hemolysin.

Figure 5

Nanopore researchers at the Advances in Genome Biology and Technology Meeting, Marco Island, Florida, 2012. Left to right: (standing) Jens Gundlach, Kristen Stoops (ONT) and M.A.; (seated) D.D. and D.B. The photograph was taken shortly after Clive Brown’s plenary seminar introducing an early prototype of the MinION sequencer developed by ONT.

Figure 6

Hagan Bayley (center) conversing with Jene Golovchenko (Harvard, left), Muthukumar Murugappan (University of Massachusetts, Amherst, back to camera) and Reza Ghadiri (Scripps Research Institute) at the April 2011 meeting of NHGRI Principal Investigators.