Oxford Nanopore Sequencing

The journey that started with the Sanger technique in 1977 on sequencing in order to decode DNA strands has witnessed the development of many techniques until today. Unlike the Sanger and Maxam-Gilbert techniques which are grouped as the first generation, next-generation sequencing techniques enable the reading of more DNA sequences in a short time. New generation sequencing techniques have grouped into second, third, and even fourth generation sequencing. It is going to be explained the basic principles and properties of Oxford Nanopore Sequencing which is one of the next-generation techniques. The technique has been grouped by third-generation or even as fourth-generation sequencing [1], [2].

Principle

There are nanopores that form a channel on a synthetic membrane. There are enzymes called unwinding enzymes inside the nanopores. At the same time, the ion flow is provided by applying a voltage to the nanopore.

When DNA isolated from any sample passes through the pore, the enzyme unwinds its double helix structure and allows the passage of a single strand.  Meanwhile, ion flow continues on the nanopore. Due to the different sizes of DNA bases which are Adenine, Guanine, Cytosine, and Thymine, it is also observed differences in the interruptions in the ion flow. These differences are recorded in real-time using the software. Then, the software gives the DNA strand sequence according to which base corresponds to which current [3].

General Properties

Oxford nanopore sequencing technology enables identifying DNA, RNA, microRNA, or proteins.

The technique provides a great advantage as it can read different lengths regardless of the length of the DNA. In second-generation sequencing techniques, DNA is divided into small pieces called fragments, and readings are made, and then the obtained DNA sequences are combined with software to obtain the base sequence of the entire DNA. However, it can be sequenced with a longer length read and a shorter time in the technique.

More increase the error rate, longer read length, and shorter time reading. For this reason, researchers use different techniques together, instead of one technique [4], [1].

Kaynaklar

[1] UESTC-Software team. (n.d.). DNA Storage Information System. Team: UESTC-software/Members. http://2016.igem.org/Team:UESTC-software/Members. 

[2] Verma, M., Kulshrestha, S., & Puri, A. (2016). Genome sequencing. Methods in Molecular Biology, 3-33. doi:10.1007/978-1-4939-6622-6_1

[3] Decoding DNA with a pocket-sized sequencer. Science in School. (n.d.). https://www.scienceinschool.org/content/decoding-dna-pocket-sized-sequencer. 

[4] Feng, Y., Zhang, Y., Ying, C., Wang, D., & Du, C. (2015). Nanopore-based fourth-generation DNA sequencing technology. Genomics, proteomics & bioinformatics, 13(1), 4-16.