Nanopore sequencing, developed by Oxford Nanopore Technologies (ONT), is a powerful method for rapidly sequencing single ultralong RNA and DNA molecules, aiding in the study of epigenomes, genomes, transcriptomes, and epitranscriptomes.1
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Introduction to Nanopore Sequencing Technology
The first DNA sequencing was performed by Sanger and co-workers in the 1970s, using a method based on the attenuation of the growing nucleotide chain with dideoxythymidine triphosphate (ddTTP). The sequences were detected radiographically using Polyacrylamide Gel Electrophoresis (PAGE).2
Over the years, advancements included using fluorescent markers and capillary array electrophoresis, but the Sanger method remained costly and less sensitive to low-level mutant alleles.
Next-generation sequencing (NGS) technologies, developed by companies like Illumina and Roche, overcame many limitations of the Sanger method.3
NGS techniques have been crucial in medical diagnosis, particularly for detecting Single Nucleotide Polymorphisms (SNPs) and their associated diseases. They also enable the study of gene expression regulation, microorganism genomes, and minimal residual disease detection.
Nanopore sequencing is a relatively recent addition to NSG technology. It is a fourth-generation method developed and commercialized by ONT.4 This technology involves passing single-strand RNA or DNA molecules through a very small protein channel (nanopore) embedded in an electrically resistant membrane.
The ion current changes as the molecules pass through, allowing the determination of the molecular sequence and identification of base modifications.
MinION was ONT’s prototype of nanopore sequencing technology, later updated to PromethION in 2015, which exhibited improved throughput.1
Principle of Nanopore Technology
The nanopore technique is based on the principles of the classical Coulter Counter or ion channels.5 The concept of nucleic acid transport through nanopores was first demonstrated using the α-hemolysin channel (biological nanopore).
Charged polymers are electrophoretically translocated through a nanometer-sized aperture or nanopores that are present in a thin membrane. The nanopore is positioned in an electrochemical chamber, separated into cis– and trans-compartments. Each compartment contains conducting buffers.6
This technique involves placing a cathode and anode on the solution’s upper/forward and lower/reverse sides of the membranes, respectively. Upon application of voltage, electrolyte ions flow through the nanopore, measured as current, typically on the pico-Ampere scale, in the electrical circuit.
Negatively charged molecules (like DNA) placed on the forward side move through the nanopores due to the electrophoretic force. The current levels of different biomolecules are measured and analyzed as they translocate through the pores.7
Key parameters for characterizing DNA translocation events include current blockage amplitude, distinctive electrical signature, event duration or dwell time, and exponential decay constant derived from the dwell time distribution. Force spectroscopy is used to analyze the physics behind the DNA translocation process.8
Nanopores Used in DNA Sequencing
DNA strands pass through nanopores, allowing for the identification of one nucleotide base at a time based on current amplitude, thereby revealing the DNA sequence.
Nanopores are broadly classified into two types: biological, which are embedded in lipid bilayers, and synthetic solid nanopores, which are fabricated on solid substrates, such as graphene, silicon nitride, and aluminum oxide.
Biological nanopores
The ideal substrates for all biological pores include planar lipid membranes, polymer membranes, or liposomes embedded in an electrochemical chamber.5 Many biological nanopores are used for nucleic acid analysis, including α-hemolysin (α-HL), cytolysin A (ClyA), aerolysin, the phi29 Motor, and Mycobacterium smegmatis porin A (MspA).9
Aerolysin is a heptameric β-pore-forming toxin with a diameter of around 1.0 nm. The α-hemolysin pore, an exotoxin secreted by Staphylococcus aureus, has a pore size of approximately 33-kDa.
This mushroom-like heptameric transmembrane pore consists of a vestibule connected to a transmembrane β-barrel, narrowing to about 1.4 nm at the vestibule.5 This pore is restricted to the translocation of ssDNA (~1 nm in diameter). The phi29 system is utilized for the passage of ssDNA, dsDNA, peptides, and small proteins.5
Synthetic solid-state nanopores
Synthetic solid-state nanopores are versatile alternatives to biological nanopores due to their unique dimensions, mechanical robustness, well-defined geometries, ease of modification, and compatibility with multiple electronic or optical measurement techniques.
The diameter of solid-state nanopores can be regulated to range from sub-nanometers to hundreds of nanometers.5
Typically, dielectric materials are used to design solid-state nanopores due to their superior thermal and chemical stability compared to lipid membranes. Graphene-based nanopores have shown considerable advantages over their biological counterparts due to their unique electrical properties.
Strengths and Limitations of Nanopore Technology for DNA Sequencing
Nanopore sequencing helps scientists identify genetic variants and provides new insights into cancer, immunology, and neuroscience-related research. It is also used to classify and monitor microbes.
Compared to traditional sequencing techniques, nanopore-based DNA sequencing offers advantages such as short processing/sequencing time, high throughput, low cost, minimal material requirements, and label-free, ultralong sequencing reads.1
Despite these advantages, there are limitations, such as context-dependent sequencing bias and low sequencing accuracy. These issues are mainly due to the lack of appropriate optical and electrical technologies to process the ultra-fast DNA passage through nanopores, detecting individual nucleotides with high sensitivity and confidence. The speed of DNA passage needs to be controlled to enable accurate base discrimination.
To reduce the DNA passage speed, scientists have employed various tactics, including using a conducting solution with increased viscosity, altering the pH of the conducting buffer, reducing applied voltage, modifying the charge distribution and size of the nanopore, and using a low melting point agarose matrix.
Although these modifications have improved read quality compared to initial nanopore sequencing reads, the signal-to-noise ratio remains a significant challenge, even with minor speed reductions.
Better methodologies are thus needed to improve the accuracy of sequence reads and fully realize the potential of nanopore technology in genomic research.
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References and Further Reading
- Lin, B., Hui, J., Mao, H. (2021). Nanopore Technology and Its Applications in Gene Sequencing. Biosensors. doi.org/10.3390/bios11070214
- Tipu HN, Shabbir A. (2015). Evolution of DNA sequencing. J Coll Physicians Surg Pak. https://pubmed.ncbi.nlm.nih.gov/25772964/
- Gupta, N., Verma, VK. (2019). Next-Generation Sequencing and Its Application: Empowering in Public Health Beyond Reality. Microbial Technology for the Welfare of Society. doi.org/10.1007/978-981-13-8844-6_15
- Feng, Y., Zhang, Y., Ying, C., Wang, D., Du, C. (2015). Nanopore-based fourth-generation DNA sequencing technology. Genomics Proteomics Bioinformatics. doi.org/10.1016/j.gpb.2015.01.009
- Haque, F., Li, J., Wu, HC., Liang, XJ., Guo, P. (2013). Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA. Nano Today. doi.org/10.1016/j.nantod.2012.12.008
- Maglia, G., Heron, AJ., Stoddart, D., Japrung, D., Bayley, H. (2011). Analysis of single nucleic acid molecules with protein nanopores. Methods Enzymol. doi.org/10.1016/S0076-6879(10)75022-9
- Timp, W., Mirsaidov, UM., Wang, D., Comer, J., Aksimentiev, A., Timp, G. (2011). Nanopore Sequencing: Electrical Measurements of the Code of Life. IEEE Trans Nanotechnol. doi.org/10.1109/TNANO.2010.2044418
- Tropini, C., Marziali, A. (2007). Multi-nanopore force spectroscopy for DNA analysis. Biophys J. doi.org/10.1529/biophysj.106.094060
- Bhatti, H., et al. (2011). Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants. RSC Adv. doi.org/10.1039/d1ra02364k
