Concept: Nanopore sequencing
This tutorial review will introduce and explore the fundamental aspects of nanopore (bio)sensing, fabrication, modification, and the emerging technologies and applications that both intrigue and inspire those working in and around the field. Although nanopores can be classified into two categories, solid-state and biological, they are essentially two sides of the same coin. For instance, both garner popularity due to their ability to confine analytes of interest to a nanoscale volume. Due to the vast diversity of nanopore platforms and applications, no single review can cover the entire landscape of published work in the field. Therefore, in this article focus will be placed on recent advancements and developments taking place in the field of solid-state nanopores. It should be stated that the intention of this tutorial review is not to cite all articles relating to solid-state nanopores, but rather to highlight recent, select developments that will hopefully benefit the new and seasoned scientist alike. Initially we begin with the fundamentals of solid-state nanopore sensing. Then the spotlight is shone on the sophisticated fabrication methods that have their origins in the semiconductor industry. One inherent advantage of solid-state nanopores is in the ease of functionalizing the surface with a range of molecules carrying functional groups. Therefore, an entire section is devoted to highlighting various chemical and bio-molecular modifications and explores how these permit the development of novel sensors with specific targets and functions. The review is completed with a discussion on novel detection strategies using nanopores. Although the most popular mode of nanopore sensing is based upon what has come to be known as ionic-current blockade sensing, there is a vast, growing literature based around exploring alternative detection techniques to further expand on the versatility of the sensors. Such techniques include optical, electronic, and force based methods. It is perhaps fair to say that these new frontiers have caused further excitement within the sensing community.
Pore me another one: Sub-nanomolar sequence-specific DNA detection and sub-micromolar small-molecule (ATP) detection was shown by way of self-assembly and disassembly of DNA superstructures within solid-state nanopores. These DNA structures provide a built-in amplification mechanism to increase the signal strength and sensitivity. This sensor was also shown to work within complex mixtures, such as mammalian serum.
Controlling DNA translocation speed is of critical importance for nanopore sequencing as free electrophoretic threading is far too rapid to resolve individual bases. A number of promising strategies have been explored in recent years, largely driven by the demands of next-generation sequencing. Engineering DNA-nanopore interactions (known to dominate translocation dynamics) with organic coatings is an attractive method as it does not require sample modification, processive enzymes, or complicated and expensive fabrication steps. In this work, we show for the first time 4-fold tuning of unfolded, single-file translocation time through small, amine-functionalized solid-state nanopores by varying the solution pH in situ. Additionally, we develop a simple analytical model based on electrostatic interactions to ex- plain this effect which will be a useful tool in designing future devices and experiments.
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.
Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. However, several applications are limited by relatively high single-read error rates (>10 %), including RNA-seq, haplotype sequencing and 16S sequencing.
The highly portable Oxford Nanopore MinION sequencer has enabled new applications of genome sequencing directly in the field. However, the MinION currently relies on a cloud computing platform, Metrichor (metrichor.com), for translating locally generated sequencing data into basecalls.
Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼10(4) nanopores per mm(2) in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording.
The accuracy of sequencing single DNA molecules with nanopores is continually improving, but de novo genome sequencing and assembly using only nanopore data remain challenging. Here we describe PoreSeq, an algorithm that identifies and corrects errors in nanopore sequencing data and improves the accuracy of de novo genome assembly with increasing coverage depth. The approach relies on modeling the possible sources of uncertainty that occur as DNA transits through the nanopore and finds the sequence that best explains multiple reads of the same region. PoreSeq increases nanopore sequencing read accuracy of M13 bacteriophage DNA from 85% to 99% at 100× coverage. We also use the algorithm to assemble Escherichia coli with 30× coverage and the λ genome at a range of coverages from 3× to 50×. Additionally, we classify sequence variants at an order of magnitude lower coverage than is possible with existing methods.
Recent advances in single molecule real-time (SMRT) and nanopore sequencing technologies have enabled high-quality assemblies from long and inaccurate reads. However, these approaches require high coverage by long reads and remain expensive. On the other hand, the inexpensive short reads technologies produce accurate but fragmented assemblies. Thus, a hybrid approach that assembles long reads (with low coverage) and short reads has a potential to generate high-quality assemblies at reduced cost.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 2 years ago
Ion transporters in Nature exhibit a wealth of complex transport properties such as voltage gating, activation, and mechanosensitive behavior. When combined, such processes result in advanced ionic machines achieving active ion transport, high selectivity, or signal processing. On the artificial side, there has been much recent progress in the design and study of transport in ionic channels, but mimicking the advanced functionalities of ion transporters remains as yet out of reach. A prerequisite is the development of ionic responses sensitive to external stimuli. In the present work, we report a counterintuitive and highly nonlinear coupling between electric and pressure-driven transport in a conical nanopore, manifesting as a strong pressure dependence of the ionic conductance. This result is at odds with standard linear response theory and is akin to a mechanical transistor functionality. We fully rationalize this behavior on the basis of the coupled electrohydrodynamics in the conical pore by extending the Poisson-Nernst-Planck-Stokes framework. The model is shown to capture the subtle mechanical balance occurring within an extended spatially charged zone in the nanopore. The pronounced sensitivity to mechanical forcing offers leads in tuning ion transport by mechanical stimuli. The results presented here provide a promising avenue for the design of tailored membrane functionalities.