Recent advances in nanopore sequencing

Abstract

The prospect of nanopores as a next-generation sequencing platform has been a topic of growing interest and considerable government-sponsored research for more than a decade. Oxford Nanopore Technologies recently announced the first commercial nanopore sequencing devices, to be made available by the end of 2012, while other companies (Life, Roche, and IBM) are also pursuing nanopore sequencing approaches. In this paper, the state of the art in nanopore sequencing is reviewed, focusing on the most recent contributions that have or promise to have next-generation sequencing commercial potential. We consider also the scalability of the circuitry to support multichannel arrays of nanopores in future sequencing devices, which is critical to commercial viability.

Figure 1. Schematic of the biological nanopore instrument with representative ssDNA and protein-bound DNA events

A) The nanopore instrument uses an amplifier to apply a command voltage V_c and measure ionic current I_p through the nanopore channel. B) At 120 mV in 1 M buffered KCl solution, 120 pA of open channel current is attenuated to ∼15 pA for ∼0.2 ms upon capture of ssDNA into the channel from the cis-chamber, until the DNA passes through the pore. With ExoI and ssDNA in the cis chamber, bound events are also observed in which the duration of the current shift is extended (∼2 ms), while the DNA is stalled in the pore channel, until ExoI is forced to dissociate from the DNA and the DNA translocates into the trans chamber (hydrolysis is here inhibited by adding EDTA to chelate Mg²⁺).

Figure 2

Varying types and geometries of nanopores (a,b) and leading configurations for sequencing (c,d). (a) Relative dimensions (approximate) of biological pores α-hemolysin and MspA. (b) Dimensions of graphene with single-atom thickness, and range of pore diameters in varying solid-state substrates. (c) The mechanism of phi29 polymerase mediated DNA translocation developed in [26] and implemented on the MspA nanopore in [27]. The motor's ability to both polymerize (at ∼40 nt/s) and unzip (at ∼2.5 nt/s) the strand is utilized to register DNA motion progress and position sensing, with the unzipping direction shown in this illustration. Though dwell times at each position are not constant, but exponentially distributed, the rates meet the requirements for DNA speed reduction [4]. (d) Functionalized electrode readers of nucleobases via 4(5)-Substituted 1-H-Imidazole-2-carboxamide. Different 180° rotations occur over the specified bonds on the carboxamide molecules to allow hydrogen bonding with different nucleobases, causing detectable variations in electron tunneling signals between two electrodes attached to the two carboxamides.

Figure 3

Tradeoff curve between die yield and cost, varying the number of patch-clamp amplifiers on the die. The optimal value is ∼820 patch-clamp amplifiers on one die, resulting in a cost of ∼$70. The actual cost is driven by the wafer cost (assumed to be $2000 here), and other factors described in the text.