DNA sequencing is the most powerful method to reveal genetic variations at the molecular level, leading to a better understanding of our body in physiological settings, and pathological conditions. It is the beginning of the long road towards better diagnostics and personalized medicine.
Even though there have been great advances in DNA sequencing technologies there are still many setbacks. These include time-consuming sample preparation, complicated algorithms for data processing, low throughput, high cost, and short read lengths. For example, the Sanger method, which is commonly used for DNA sequencing, typically requires two working days, a PCR step and a very high quantity of nucleic acid fragments to produce a detectable band by gel electrophoresis! It is safe to say that there are still some adjustments to be made.
Sanger sequencing is considered to be the first generation of sequencing methods; amplification-based massively parallel sequencing is the second generation; and single-molecule sequencing is the third generation.
After the development of three generations, DNA sequencing technology is now entering the era of single-molecule nanopore sequencing technology – the fourth generation.
What Is Nanopore Sequencing?
The concept is that if each base could produce different ionic current torrents during DNA translocating through a small channel (nanopore), then it would be possible to distinguish between different nucleotides. The pores are usually in a biological membrane, or in a solid-state film, that separates two compartments that contain conductive electrolytes. Electrodes are immersed in each compartment. The resulting electric field causes the electrolyte ions in solution to move through the pore through electrophoresis, generating an ionic current signal. When the pore is blocked, due to the passage of a biomolecule, the current flow is also blocked. You can determine the physical and chemical properties of the target molecules by analyzing the amplitude and duration of the blockades.
In sequencing, each nucleotide blocks the channel differently giving a different amplitude and duration of the blockade. This information is converted into DNA sequence information.
Nanopore technologies are broadly divided into two categories: biological and solid-state.
A nanopore-based diagnostic tool offers various advantages:
- it detects target molecules at very low concentrations;
- it screens panels of biomarkers or genes;
- provides a fast analysis at low cost;
- and, finally, it eliminates cumbersome amplification and conversion steps, that may introduce biases and errors.
Nanopore sequencing has the potential to be a great tool: both in the genomic, and epigenomic department.
This technique can be used for cancer diagnosis and treatment through the detection and accurate quantification of MicroRNA (miRNA – cancer biomarkers) and the detection of aberrant DNA methylation, which can serve as a robust biomarker in cancer. Therefore, itcan be a great asset in an oncology setting by facilitating cancer diagnosis, staging, progression, prognosis and treatment response.
Since nanopore sequencing produces fast results it is a powerful tool for the diagnosis of infectious agents. Truly, the advantages in read length, and time to pathogen identification may be crucial in a hospital environment, where time may mean the difference between a successful treatment and death.
Indeed, MinION (a portable device manufactured by Oxford Nanopore Technologies) successfully analyzed Ebola samples on-site in Africa. Astonishingly, the confirmation of Ebola sequences took as little as 15 minutes of MinION run time! The great part about it? It’s not even necessary a full on lab to do this experiment – which means that on-site experiments just got a whole lot more on-site.
This All Seems Very Good… What Is the Catch?
The significant advantages of nanopores include label-free, ultra-long reads, high throughput, low material requirement and it doesn’t suffer from amplification biases introduced by PCR. Each of these greatly simplifies the experimental process and can be easily used for DNA sequencing applications.
However, there is a catch. More specifically, there are still two significant challenges to overcome: very high speed of the translocation and low signal sensitivity. DNA molecules translocate through the channels with a speed of approximately 1 base/μs, which is too high for reliable detection of different nucleotides. This makes it difficult to distinguish between the four nucleotides, leading to low sensitivity. Additionally, although there are already some great applications for nanopore sequencing, it is prone to error. It still has a higher error rate than current biochemical sequencing platforms; making it a bad choice for thorough sequencing, such as SNP sequencing. And, although there is still room for improvement, nanopore sequencing is improving day-by-day. It is just a matter of time before we truly enter the era of the fourth generation of sequencing methods!