The process involved in DNA sequencing is of figuring out the order of DNA bases. It applies to the human genome that allows scientists to sequence genes and genomes. DNA sequencing allows scientists to make accurate predictions about an endless number of areas such as genetic diseases, cancer, and immune systems and further how it will affect individuals based on their genetic profiles. It is the most powerful method to reveal genetic variations at the molecular level such as single nucleotide polymorphism, copy number variation, gene fusion, and insertion or deletion which are relevant to genetic diseases. Current sequencing methods are time-consuming and quite expensive and are still not well optimized from the point of view of cost and speed. With expanding research, there is an increasing demand for advanced sequencing technologies. One of the most promising new approaches to DNA sequencing, that is cost-effective and accurate, is the use of biopolymer translocation via nanopores known as nanopore sequencing. Nanopore-based sequencing is a complex process that utilizes both experimental and theoretical methods to determine the sequence of nucleic acids which is inferred from the changes in the ionic current across a membrane when a single DNA molecule passes through a protein nanopore. This nanopore sequencer uses a label-free and amplification-free single-molecule approach which can sequence an ultra‐long read limited by the input nucleotide length or determine the real-time analysis of long DNA or RNA fragments. The signal received is further decoded to present the unambiguous DNA or RNA sequence. Nanopore-based sequencing is used to-
- Resolve complex structural variants and repetitive regions
- Simplify de novo genome assembly
- Improve existing reference genomes
- Study linkage and phasing
- Enhance metagenomic identification of closely related species
- Distinguish plasmid from genome
- Sequence entire microbes in single reads
- Explore epigenetic modifications
Several sensing mechanisms have been proposed and explored in recent years such as ionic current blockades, transverse current measurements of DNA translocation, and optical recognition. Nanopore technologies open a new dimension to molecular biology exploration and can be broadly alienated into two categories which are biological and artificial pores. In recent years, many fields such as medicine, epidemiology, ecology, and education have benefited from this technology. The advantages of nanopore sequencing are that it is faster, more efficient, cost-effective, direct procedure, minimal requirements for chemical modifications, or enzyme-dependent amplifications. This technology also eliminates the need for additional enzymes such as polymerases or ligases and the purified fluorescent reagents. There are several problems associated with this technology such as its rapid DNA translocation velocity (∼1–3 μs/base), which is too high for reliable detection of different nucleotides, increasing sensitivity and chemical specificity.
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