Oxford Nanopore Sequencing: Principle, Protocol, Uses microbiologystudy

Oxford Nanopore Sequencing is a nanopore sequencing method developed by Oxford Nanopore Technologies (ONT) that identifies the sequence of nucleotides by passing individual molecules through nanoscale pores and measuring changes in the electrical current.

The nanopores only allow the passage of single-stranded DNA in a linear sequence. This method provides real-time analysis and can sequence nucleotides from short to ultra-long reads.

Single-molecule detection methods like nanopore sequencing are included in the third generation of sequencing technologies. The concept of nanopore sequencing was developed in the 1980s while the first nanopore sequencing instrument was developed by ONT in the 2010s. ONT’s devices have made nanopore sequencing widely available. It is used in genomic studies, pathogen identification, epigenetic analysis, and various other fields.

Historical Background of Oxford Nanopore Sequencing

• In 1989, Professor David Deamer first came up with the idea of using a protein channel in membranes to detect individual nucleotides. 
• In 1991, Deamer discussed this idea with Professor Dan Branton from Harvard which led to the first collaborative research efforts in nanopore sensing.
• Initial experiments with nanopore sensing began in 1993 with Professor George Church joining the team along with several other researchers. The concept of nanopore sequencing was published in 1996.
• In 2001, Professor Hagan Bayley at Oxford University explained a working nanopore sensor. This led to the establishment of Oxford Nanopore Technologies in 2005 by Professor Bayley along with Dr. Gordon Sanghera and Dr. Spike Willcocks.
• The first nanopore sequencing data was presented in 2012 at a conference, introducing the MinION and GridION systems.
• MinION was launched for access to early users through the MinION Access Programme (MAP) in 2014 and it was commercially released in 2015.
• In 2016, new devices like the mobile-compatible SmidgION and the automated sample preparation device VolTRAX were announced.
• In 2017, the company launched the GridION X5 while the PromethION was commercially available in 2018.
• In 2019, the Flongle adapter was launched for low-cost, smaller sequencing tests.
• The company announced the launch of PromethION 2 in 2022.

Principle of Oxford Nanopore Sequencing

Nanopore sequencing works on the principle that as nucleic acid molecules pass through a nanopore channel in a membrane that separates two electrolyte-filled chambers, it disrupts the current and produces a characteristic electrical signal. The chamber where sequencing occurs is called the cis side, while the chamber containing the analyte is the trans side. There are motor proteins that control the speed of translocation. These proteins also have helicase activity which unwinds the double-stranded DNA into single-stranded molecule. When voltage is applied across the membrane, it generates an ionic current. As nucleotides pass through the nanopore, their negative charge causes them to move toward the anode, which disrupts the ionic current and generates a distinct pattern. Different nucleotides affect the ionic current differently and produce unique patterns due to their mass and electrical properties. This pattern is detected and interpreted to determine the nucleotide sequence.

Video on Principle of Oxford Nanopore Sequencing

Process of Oxford Nanopore Sequencing

1. DNA Extraction and Library Preparation

• The initial step involves extracting the genetic material of interest from different samples.
• For ultra-long reads, special experimental methods are used to isolate high molecular weight DNA. Different methods like spin column, magnetic bead, and phenol-chloroform extraction are used to extract the DNA.
• The extracted genetic material may be fragmented into smaller pieces for sequencing using physical shearing methods or enzymatic digestion.
• The fragmented DNA can undergo optional size selection to isolate fragments of specific lengths.
• The fragmented DNA is then repaired to ensure that the ends of the DNA fragments are suitable for accurate sequencing.
• Adaptors are added to the ends of the DNA fragments which help in attaching the DNA fragments to the motor proteins and nanopores. 

2. Sequencing Process

• The library is introduced into a flow cell for sequencing. It contains two ionic solution-filled chambers separated by a membrane containing nanopores.
• The flow cell is placed within a sequencer where a constant voltage is applied. This creates an ionic current through the nanopores.
• The DNA to be sequenced is mixed with motor proteins that bind to the DNA. The motor protein unwinds the double helix and transports one strand through the nanopore.
• When the single-stranded DNA moves through the nanopore, it interferes with the ionic current. Each nucleotide causes a specific change in the current generating specific signals. This signal is detected by a patch-clamp amplifier.

3. Data Analysis

• The signals are translated into DNA sequences using base-calling algorithms that convert the raw current measurements into nucleotide sequences. This involves interpreting the specific patterns of current changes. DNA or RNA modifications are also detected using base calling.
• After base calling, error correction is done to refine the sequence data. This corrects errors in the sequence to improve the accuracy of sequencing data.
• The sequenced data is aligned to a reference genome and genome assembly is performed. 
• Structural variants and repetitive regions are detected following assembly and alignment. 
• For transcriptome studies, full-length gene isoforms are reconstructed, and gene expression is studied.

Types of Oxford Nanopore Sequencing

There are three methods of nanopore sequencing:

a. 2D Sequencing

In 2D sequencing, both the template and the complementary strand are sequenced. 2D sequencing was one of the initial methods used by ONT. A hairpin adaptor is used to link the two strands, allowing the sequencing of the template strand first followed by the complementary strand. This method provides higher accuracy since it sequences both strands of DNA. However, as of May 2017, ONT has phased out 2D flow cells. 

b. 1D Sequencing

In 1D sequencing, only the template DNA strand is sequenced. The DNA is independently ligated with an adapter and passed through the nanopore for sequencing. This method is simple and faster than 2D sequencing but is less accurate and provides less detailed information compared to other methods. 

c. 1D² Sequencing

The 1D² method builds on the principles of 2D sequencing but does not use hairpins to connect the strands. It sequences only one strand at a time but uses special adapters that allow the sequencing of both strands independently in separate passes through the nanopore. So, it sequences both the template and complementary strands independently increasing the accuracy of the reads.

Types of Nanopores

Nanopores used in sequencing can be derived from biological sources or synthesized from solid-state materials. 

1. Biological nanopores

Biological nanopores are derived from natural proteins. They are generally produced by microorganisms. These include membrane proteins like α-hemolysin from Staphylococcus aureus and Mycobacterium smegmatis porin A (MspA) protein. These biological pores naturally form channels that can be used for DNA sequencing. These nanopores have a shorter lifespan and are less stable compared to solid-state nanopores.



2. Solid-state nanopores

Solid-state nanopores or synthetic nanopores are made from solid-state materials such as silicon nitride, aluminum oxide, or carbon nanotubes. Synthetic nanopores can be designed to the exact requirements needed for sequencing. They are created using various methods that allow control over the properties of nanopores.

Oxford Nanopore Sequencing Devices

1. MinION

MinION is a portable and compact Oxford Nanopore sequencing device. It is the first nanopore sequencing instrument introduced in 2012 and commercially launched in 2015. The MinION can sequence any length of fragments, from short to ultra-long. It connects easily to any computer through a standard USB 3.0 cable. This device is suitable for different applications including whole genomes, metagenomics, targeted sequencing, and transcriptome analysis.



2. GridION

GridION is a compact benchtop sequencing device that can run 5 MinION flow cells simultaneously. It was introduced along with MinION in 2012 and was launched in 2017. This instrument allows multiple sequencing experiments to be conducted simultaneously. GridION also supports read lengths from short to ultra-long sequences and efficiently handles large numbers of samples.



3. PromethION

PromethION is a high-throughput device suitable for large-scale projects. It features 24 or 48 parallel flow cells. It was introduced in 2014 and commercially launched in 2018. This system supports a wide range of sequencing applications from small-scale projects to population-scale studies.



4. Flongle

Flongle is an adapter for MinION or GridION that was launched in 2019. It is suitable for smaller sequencing experiments. Flongle allows users to run single samples as needed without the need for multiplexing. It has applications in amplicon sequencing, quality control, and other small sequencing tests.



Advantages of Oxford Nanopore Sequencing

• Oxford Nanopore sequencing provides real-time data and it can be scaled to any device size which is suitable for different experimental needs. 
• Oxford Nanopore sequencing devices like MinION are compact and portable which allows sequencing even outside traditional lab environments. 
• It also contains devices that allow rapid library preparation which speeds up the sequencing process.
• Different parameters in Oxford Nanopore sequencing like experiment duration, device used, and the number of flow cells used can be adjusted to specific research needs.
• Oxford Nanopore sequencing devices can sequence long fragments of DNA supporting ultra-long read lengths. Long reads simplify genome assembly.
• Oxford Nanopore sequencing can directly sequence native DNA or RNA without PCR amplification which reduces bias.

Limitations of Oxford Nanopore Sequencing

• Nanopore sequencing has a higher error rate compared to other sequencing technologies due to the difficulties in accurately interpreting the ionic current disruptions caused by nucleotides passing through the nanopore. 
• Another limitation of Oxford Nanopore sequencing is the need for large amounts of input nucleic acid material.
• Nanopore sequencing data can be complex to analyze. Advanced bioinformatics tools and computational resources are required to accurately interpret the data. 
• It has lower throughput which limits its applications in large-scale studies.
• Nanopore sequencing can be sensitive to sample preparation and contamination. Producing ultra-long reads consistently can be difficult and factors like DNA quality may affect the read lengths.

Applications of Oxford Nanopore Sequencing

• The long-read capability of Oxford Nanopore sequencing helps in the identification and study of genetic variants and novel isoforms. This is useful in understanding complex genetic conditions and diseases.
• Nanopore sequencing is used to study structural variation in cancer genomes which is important for diagnosing and monitoring cancer. 
• Nanopore sequencing can be used in the identification of microorganisms. This helps in the classification and monitoring of microbes. It is also used in pathogen detection and antibiotic resistance profiling to identify resistance genes from clinical samples and to monitor outbreaks of infectious diseases.
• Nanopore sequencing can be used to assemble highly repetitive regions and structural variations leading to more complete genome assemblies.
• Nanopore sequencing can detect DNA modifications like methylation patterns which is useful for understanding epigenetic modifications.
• ONT devices like MinION are portable which allows on-site research such as identifying microbes in polluted rivers and pests in agricultural fields.

References

1. Advantages of nanopore sequencing. (n.d.). Retrieved from https://nanoporetech.com/platform/technology/advantages-of-nanopore-sequencing
2. Delahaye, C., & Nicolas, J. (2021). Sequencing DNA with nanopores: Troubles and biases. PLoS ONE, 16(10), e0257521. https://doi.org/10.1371/journal.pone.0257521
3. Heather, J. M., & Chain, B. (2016). The sequence of sequencers: The history of sequencing DNA. Genomics, 107(1), 1–8. https://doi.org/10.1016/j.ygeno.2015.11.003
4. How nanopore sequencing works | Oxford Nanopore Technologies. (n.d.). Retrieved from https://nanoporetech.com/platform/technology
5. Jain, M., Olsen, H. E., Paten, B., & Akeson, M. (2016). The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biology, 17(1). https://doi.org/10.1186/s13059-016-1103-0
6. Lin, B., Hui, J., & Mao, H. (2021). Nanopore Technology and Its Applications in Gene Sequencing. Biosensors, 11(7), 214. https://doi.org/10.3390/bios11070214
7. MacKenzie, M., & Argyropoulos, C. (2023). An Introduction to Nanopore Sequencing: Past, Present, and Future Considerations. Micromachines, 14(2), 459. https://doi.org/10.3390/mi14020459
8. Nanopore Sequencing: Principles, Platforms and Advantages – CD Genomics. (n.d.). Retrieved from https://www.cd-genomics.com/nanopore-sequencing-principles-platforms-and-advantages.html
9. Oxford Nanopore Technologies. (n.d.). Retrieved from https://nanoporetech.com/about/history
10. Preul, M., Patel, A., Belykh, E., Miller, E., George, L., Martirosyan, N., & Byvaltsev, V. (2018). MinION rapid sequencing: Review of potential applications in neurosurgery. Surgical Neurology International, 9(1), 157. https://doi.org/10.4103/sni.sni_55_18
11. Wang, Y., Zhao, Y., Bollas, A., Wang, Y., & Au, K. F. (2021). Nanopore sequencing technology, bioinformatics and applications. Nature Biotechnology, 39(11), 1348–1365. https://doi.org/10.1038/s41587-021-01108-x