Nanopore Sequencing

Nanopore sequencing is a modern "third generation" DNA sequencing technology that differs fundamentally from Illumina or other NGS methods. The method enables the direct reading of individual DNA or RNA molecules in real time, without prior amplification (duplication). It was mainly developed by the company Oxford Nanopore Technologies (ONT).

Basic principle of nanopore sequencing: In this method, a single-stranded DNA or RNA is passed through a tiny nanopore (a biological or synthetic "molecular opening") in a membrane. As the molecule passes through, it alters the ionic current flowing through the pore. These current changes are characteristic of each base (A, T, C, G) and can be measured electronically.

1. sample preparation: The DNA or RNA is combined with a motor enzyme that pulls the molecule through the pore in a controlled manner. In contrast to Illumina sequencing, no complex amplification or labeling steps are necessary.

2nd passage through the nanopore: The nanopore is located in an electrically conductive membrane. A voltage is applied and ions flow through the pore, generating an electric current. When DNA or RNA slides through the pore, the bases interfere with the current to varying degrees. Each base motif (e.g. 5-Mer) generates a unique current signal.

3. signal measurement and base calling: The current is measured continuously. Software converts the current patterns into base sequences (A, T, C, G). This process takes place in real time - you can see the sequence "live" while it is being read.

Rapid advances in nanopore technology for sequencing single long DNA and RNA molecules have led to significant improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of experimental and bioinformatics methods to fully exploit the long read lengths of nanopores for the study of genomes, transcriptomes, epigenomes and epitranscriptomes (McCormick CA et al. 2024).

However, analyzing the raw data of nanopore signals offers far more possibilities than simply sequencing genomes and transcriptomes: algorithms that use machine learning to extract biological information from these signals enable the detection of DNA and RNA modifications, the estimation of poly(A) tail length and the prediction of RNA secondary structures (Wan YK et al. 2022).

In bacterial gene expression, nanopore direct RNA sequencing (DRS) provides a promising platform for rapid and comprehensive characterization of bacterial RNA biology. Using unmodified in vitro transcribed (IVT) RNA libraries as negative controls, several Nanopore-based computational tools can identify putative modification sites in the transcriptomes of E. coli and S. aureus. In combination with next-generation sequencing-based N6-methyladenosine (m6A) detection methods, 75 highly reliable m6A candidates were identified in the protein-coding transcripts of E. coli, while none were detected in S. aureus. This reveals the potential of Nanopore DRS for systematic and convenient transcriptome and epitranscriptome analysis (Tan L et al. 2024).

1. Hong A et al. (2022) Analyzing viral epitranscriptomes using nanopore direct RNA sequencing. J Microbiol 60:867-876.
2. McCormick CA et al. (2024) Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis. bioRxiv 28:2023.04.06.535889.
3. Tan L et al. (2024) Analysis of bacterial transcriptome and epitranscriptome using nanopore direct RNA sequencing. Nucleic Acids Res 52:8746-8762.
4. Wang Y et al. (2021) Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol 39:1348-1365.
5. Wan YK et al. (2022) Beyond sequencing: machine learning algorithms extract biology hidden in Nanopore signal data. Trends Genet 38:246-257.