Introduction:
- Nanopore sequencing is a method of DNA sequencing that uses nanopore technology to read DNA molecules as they pass through a nanopore, or small hole, in a membrane. This technology allows for rapid, real-time sequencing of DNA or RNA molecules, and has several advantages over other DNA sequencing methods.
- Nanopore sequencing is considered to be a fourth-generation DNA sequencing technology. This approach allows for real-time, high-throughput sequencing of DNA or RNA molecules with long reads and low error rates.
- Oxford Nanopore Technologies Ltd. discovered nanopore sequencing technology, which represents the most powerful way for rapidly generating long-read sequences. Nanopore sequencing can sequence a single molecule of DNA or RNA without the requirement for PCR amplification or chemical labeling of the sample.
- One advantage of nanopore sequencing is its high speed and throughput, which allows for the rapid sequencing of long DNA molecules. It can also be used for the sequencing of multiple DNA samples in parallel, making it a useful tool for high-throughput sequencing applications.
- Another advantage of nanopore sequencing is its ability to produce long, contiguous reads of DNA, which can be useful for applications such as genome assembly or the identification of structural variations in the genome.
- Nanopore sequencing is also relatively inexpensive compared to other DNA sequencing methods, and it does not require the use of enzymes or other reagents, making it a cost-effective option for many research and clinical applications.
- The Oxford Nanopore Platform is a DNA sequencing platform developed by Oxford Nanopore Technologies (ONT). It is based on the use of protein nanopores to sequence DNA, and it is known for its ability to generate long reads of DNA sequence data.
- The Oxford Nanopore Platform consists of several instruments, including the MinION, GridION, and PromethION. These instruments use a portable, handheld device to perform nanopore sequencing, and they are capable of generating thousands of sequences reads in a single run. The platform also includes software and analysis tools for data processing and interpretation.
Principle:
Nanopore sequencing is a method of sequencing that uses a single pore in a nanopore membrane to determine the sequence of nucleotides in a sample of DNA or RNA molecules. The molecule is passed through the pore, and the nucleotides are detected as they pass through the pore by measuring changes in an ionic current.
The method employs a nanoscale protein pore, or ‘nanopore,’ which functions as a biosensor and is placed in an electrically resistant polymer membrane. A continuous voltage is given to an electrolytic solution to generate an ionic current through the nanopore, driving negatively charged single-stranded DNA or RNA molecules through the nanopore.
A motor protein that moves the nucleic acid molecule from the negatively charged ‘cis’ side to the positively charged ‘trans’ side through the nanopore regulates the rate of translocation. As the molecules pass through the nanopore, they disrupt the electrical current flowing through the pore, and this disruption is detected by a sensor.
Fig: Principle of Nanopore sequencing
Real-time sequencing of individual molecules is possible due to changes in ionic current during translocation that are related to the nucleotide sequence found in the sensing area and are decoded using computer algorithms. By analyzing the pattern of current disruptions, the sequence of the DNA or RNA molecules can be determined.
The principle behind nanopore sequencing is based on the fact that each nucleotide has a unique size and charge, which causes distinct changes in the ionic current as it passes through the nanopore, or small hole. By measuring these changes in the ionic current, it is possible to determine the sequence of nucleotides in the DNA sample.
There are several different types of nanopore sequencing technologies, but one common approach is to use an enzyme called helicase to unwind the DNA double helix into a single strand. The single-stranded DNA molecule is then passed through a nanopore, and the sequence is read as the molecule moves through the pore.
Steps:
There are several steps involved in nanopore sequencing:
Sample preparation: The first step in nanopore sequencing is to prepare the sample of DNA for analysis. This typically involves extracting the DNA from the sample and purifying it to remove contaminants.
Library preparation: Next, the purified DNA sample is processed to create a library of DNA fragments that are suitable for nanopore sequencing. This typically involves cutting the DNA into shorter fragments and attaching adapters to the ends of the fragments.
Loading the DNA onto the flow cell: The flow cell is a device that contains the nanopore membrane and is used to perform the sequencing. The DNA fragments are loaded onto the flow cell, where they are immobilized and ready for sequencing.
Sequencing: During sequencing, the DNA fragments are passed through the nanopore one at a time, and the ionic current is measured as each nucleotide passes through the pore. The sequence of nucleotides is determined based on the changes in the ionic current.
Data analysis: After the sequencing is complete, the data is analyzed to determine the sequence of nucleotides in the DNA sample. This typically involves using computational tools to align the sequence reads and identify any errors or ambiguities.
Data interpretation: The final step in nanopore sequencing is to interpret the data and use it to answer the research question or make a diagnosis. This may involve using the sequence data to identify genetic variations, predict protein sequences, or perform other types of analyses.
Nanopore DNA sequencing devices:
MinION: This is a portable, handheld device developed by Oxford Nanopore Technologies (ONT). It is capable of generating thousands of sequences reads in a single run and is suitable for a wide range of applications, including genetic diagnosis, whole genome sequencing, and RNA sequencing.
Fig: MinION Sequencer
GridION: This is a benchtop device developed by ONT that is capable of generating tens of thousands of sequences reads in a single run. It is suitable for high-throughput applications, such as whole genome sequencing and metagenomics.
PromethION: This is a scalable, high-throughput device developed by ONT that is capable of generating millions of sequences reads in a single run. It is suitable for large-scale projects, such as whole genome sequencing and epigenomics.
Applications:
Genetic diagnosis: Nanopore sequencing can be used to identify genetic variations and mutations that may be associated with inherited diseases or conditions. It is particularly useful for identifying rare and complex genetic variations that may be missed by other DNA sequencing methods.
Whole genome sequencing: Nanopore sequencing can be used to determine the complete DNA sequence of an organism’s genome. This can provide valuable insights into the genetic basis of traits and diseases, and it can be used to study evolution and biodiversity.
Fig: SARS-CoV-2 Genome Sequencing using Oxford Nanopore Technologies
Structural variation analysis: Nanopore sequencing can be used to identify large-scale structural variations in the genome, such as insertions, deletions, and translocations. This can be useful for understanding the genetic basis of complex traits and diseases.
RNA sequencing: Nanopore sequencing can be used to study the expression of genes by sequencing RNA molecules. This can provide insights into the functions of genes and the regulation of gene expression.
Metagenomics: Nanopore sequencing can be used to study the DNA of microbial communities, such as the microbiome. This can provide insights into the diversity and functions of microorganisms in different environments.
Epigenomics: Nanopore sequencing can be used to study epigenetic modifications, such as methylation, that can affect gene expression. This can provide insights into the role of epigenetics in development and disease.
Limitations:
Accuracy: While nanopore sequencing is generally accurate, it has a higher error rate compared to some other DNA sequencing methods. This can make it more difficult to identify rare or complex genetic variations, and it may require additional data processing or analysis to correct errors.
Sensitivity: Nanopore sequencing is less sensitive than some other DNA sequencing methods, meaning that it may not be able to detect low levels of DNA or certain types of genetic variations.
Interference: Nanopore sequencing can be affected by interference from other molecules in the sample, such as proteins or RNA. This can lead to false positives or errors in the sequence data.
Cost: While nanopore sequencing is generally less expensive than other DNA sequencing methods, it is still relatively expensive and may not be practical for all research or diagnostic applications.
Complexity: Nanopore sequencing is a complex process that requires specialized equipment and trained personnel to perform. It may not be suitable for all research or diagnostic laboratories.
References:
- Bharagava, R.N., Purchase, D., Saxena, G. and Mulla, S.I., 2019. Applications of metagenomics in microbial bioremediation of pollutants: from genomics to environmental cleanup. In Microbial diversity in the genomic era (pp. 459-477). Academic Press.
- Wang, Y., Zhao, Y., Bollas, A., Wang, Y. and Au, K.F., 2021. Nanopore sequencing technology, bioinformatics and applications. Nature biotechnology, 39(11), pp.1348-1365.
- Vilgis, S. and Deigner, H.P., 2018. Sequencing in precision medicine. In Precision medicine (pp. 79-101). Academic Press.
- https://nanoporetech.com/