What is Nanopore Sequencing Technology: Uses, Benefits & Trends

Nanopore sequencing addresses the need for fast, accessible, and cost-effective genomic analysis. Enabling the real-time, portable sequencing of DNA and RNA revolutionizes research in biology and environmental science. Here we explore its core principles, diverse applications, key advantages like long-read sequencing, and the challenges that must be overcome for further optimization.

What is Nanopore Sequencing Technology?

Nanopore sequencing is a fourth-generation sequencing method that enables direct sequencing of single molecules of DNA or RNA by passing them through a tiny protein channel known as a nanopore. This nanopore, typically composed of transmembrane cellular proteins, has an internal diameter of about 1 nanometer. As the DNA or RNA strand passes through the nanopore, it temporarily obstructs the channel, causing distinctive changes in ion currents. 

These changes are unique for each nucleotide, allowing researchers to accurately determine the sequence and detect chemical modifications within the molecule. This technique supports label-free sequencing, meaning it does not require chemical labeling or PCR amplification, streamlining the sequencing process significantly.

Nanopore sequencing has evolved beyond nanopore DNA sequencing and nanopore RNA sequencing, with recent developments also pointing towards the potential for nanopore protein sequencing and broader applications in nanopore gene sequencing.

What is Oxford Nanopore and ONT Sequencing?

Oxford Nanopore Technologies (ONT), a spin-off company from the University of Oxford, has played a pivotal role in the evolution and commercialization of nanopore sequencing technology.

ONT introduced groundbreaking sequencing devices, ranging from portable, handheld sequencers like MinION—ideal for real-time field analysis—to desktop and high-throughput sequencing platforms such as GridION and PromethION, respectively. ONT continues to innovate, with ongoing research aiming to enhance sequencing accuracy, speed, and portability, making genomic sequencing more accessible and widely used across fields like clinical diagnostics, environmental monitoring, and personalized medicine.

What are Nanopores Used for?

Nanopores are transforming the way scientists analyze molecules by enabling real-time, single-molecule detection. Let’s look at some of its applications:

Direct DNA and RNA Sequencing

Nanopores allow for the sequencing of DNA and RNA without PCR amplification or chemical labeling, facilitating simpler genomic studies. This eliminates PCR bias, preserves base modifications, and provides more accurate insights into epigenetic changes and RNA transcript variations. It is particularly valuable for analyzing complex genomic regions, transcriptomic profiling, and identifying splice variants in RNA sequencing.

Genotyping

Nanopore sequencing provides a relatively inexpensive method for genotyping, making genetic analysis accessible to a wider range of applications. Its affordability and speed make it particularly beneficial for population genomics, evolutionary biology, and personalized medicine.

Characterization of Plasmids and Resistomes

This technology is effective for characterizing carbapenemase-encoding plasmids in bacteria from wastewater treatment plants, enabling detailed analysis of antibiotic resistance genes and their potential spread in environmental settings. Nanopore sequencing helps researchers correlate resistome phenotypes and genotypes, crucial for managing antibiotic resistance in public health.

Detection of Epigenetic Modifications

Nanopore sequencing can detect important epigenetic modifications, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and even DNA damage sites like abasic sites. The latest nanopore models also detect modifications like N6-methyladenine (6mA), significantly enhancing research capabilities in epigenetics, developmental biology, and cancer diagnostics.

Benefits of Nanopore Sequencing Technology

​Nanopore sequencing technology offers several notable advantages over traditional sequencing methods, making it a valuable tool.

Long-Read Sequencing

Nanopore sequencing is capable of reading exceptionally long DNA fragments, ranging from thousands to even millions of nucleotides in a single read. This ultralong-read capability (10⁴–10⁶ bases) enables researchers to analyze complex genomic regions, such as repetitive sequences, high GC content regions, telomeres, and structural variations. By providing a more comprehensive understanding of genomes, transcriptomes, and epigenomes, it is particularly useful in resolving large-scale genome assemblies and studying non-coding DNA regions.

Real-Time Results

Nanopore sequencing enables real-time analysis of genomic data. The sequencing process can achieve speeds of hundreds of kilobases per second, with each DNA strand passing through the nanopore at a rate of approximately 250 to 450 bases per second. This rapid processing allows for immediate data interpretation, making it invaluable for time-sensitive applications such as outbreak monitoring, infectious disease tracking, and clinical diagnostics.

Mobility

One of the most notable advantages of nanopore sequencing is its portability. Devices like the MinION are compact and lightweight, making them highly mobile and suitable for point-of-care applications, field research, and environmental testing. This mobility has been particularly beneficial for tracking disease outbreaks and performing real-time genomic surveillance in remote locations.

Cost-Effectiveness

Nanopore sequencing is relatively cost-effective compared to traditional sequencing platforms. It offers low-cost genotyping and has the potential for affordable sequencing, with some systems delivering sequencing at approximately $25 to $40 per gigabase of sequence. Also, nanopore technology requires minimal sample preparation, reducing reagent costs and making sequencing more accessible to a wider range of researchers and institutions.

Challenges of Nanopore Sequencing Technology

Despite its advantages, nanopore technology still faces significant challenges that hinder its widespread adoption, including accuracy issues, high error rates, etc. Here are the major issues that still need to be addressed.

Higher Error Rates Compared to Other Methods

One significant challenge is the technology's inherent error rate, which has historically been higher compared to other sequencing methods. Early nanopore sequencing efforts faced difficulties in achieving single-nucleotide resolution due to the rapid translocation speed of DNA strands through the nanopore, leading to recording challenges and background noise. Although advancements have been made to improve accuracy, achieving consistently low error rates remains an ongoing endeavor.

Unstable DNA Passage

Controlling the speed at which DNA molecules pass through the nanopore is crucial for accurate sequencing. The rapid movement of nucleotides—occurring in microseconds—poses challenges in recording precise measurements. Strategies such as protein engineering and the development of molecular motors have been employed to regulate this translocation speed, but achieving optimal control continues to be a technical hurdle.

Inconsistent Pore Dimensions

Maintaining a precisely defined and uniform size of the nanopores is a significant challenge. To enhance sequencing accuracy and resolution, both the lateral and vertical dimensions (pore diameter and length, respectively) should be as small as possible. Variations in pore size can impact the consistency of electrical signal detection, potentially reducing sequencing precision.

From Nanopore to Bio-FET: Unlocking New Possibilities in Biosensing with Molsentech

Nanopore sequencing has paved the way for transformative advances in genomics, offering unprecedented portability, real-time data acquisition, and long-read sequencing capabilities. Despite its transformative impact, nanopore sequencing still faces several challenges. Issues such as maintaining consistent pore size, optimizing signal accuracy, and improving sequencing error rates continue to hinder widespread adoption.

Recognizing these limitations, companies like Molsentech have explored alternative biosensing methods, with a strong focus on nanowire-based field-effect transistor (FET) biosensors, pushing the boundaries of what is possible in real-time molecular detection.

With a mission to revolutionize diagnostics, Molsentech has developed an advanced Bio-FET platform that integrates semiconductor biochip design, biorecognition element modification, system integration, and AI-driven analysis. Our BioFET chips have already demonstrated exceptional sensitivity and rapid detection capabilities, proving their value during the COVID-19 pandemic. Now, we are unlocking new possibilities for detecting Alzheimer’s, cancer, and infectious diseases with unmatched precision and efficiency.

Be part of the future of molecular diagnostics with Molsentech. Our cutting-edge Bio-FET chips are transforming biosensing, enabling real-time, ultra-sensitive detection for a wide range of applications. Contact us today to explore how our semiconductor biochip technology can elevate your research and diagnostic capabilities.