Nanopore sequencing technology is one of the newest sequence technology, in this technology single DNA molecules are sequenced directly, without PCR amplification step or a chemical labelling step / Library prepartion step. It offers the potential of ultra long read lengths of hundred kilobases (>100Kb). Nanopore devices perform sequencing in real time. DNA/RNA sequencing done directly. The technology is scalable from small throughput to high-throughput applications.
Nanopore sequencing is the method of the third generation sequencing. The nanopore is a tiny bio pore with a diameter of nanoscale, which can be found in protein channel embedded in lipid bilayer which facilitates ion exchange. Any particle movement can disrupt the voltage across the channel. The core concept of nanopore sequencing involves putting a thread of single-stranded DNA across α-haemolysin (αHL) pore. αHL, a 33 kD protein isolated from Staphylococcus aureus, undergoes self-assembly to form a heptameric transmembrane channel. It can tolerate extraordinary voltage up to 100mV with current 100 pA. This unique property supports a building block of the nanopore. In nanopore sequencing technology, an ionic flow is applied continuously. The Standard electrophysiological technique detects the current disruption in this technology. Readout relies on the size difference between all deoxyribonucleoside monophosphate (dNMP). Thus, for given dNMP, characteristic current modulation is shown for discrimination. Ionic current is resumed after trapped nucleotide entirely squeezing out. Nanopore sequencing possesses many fruitful advantages over existing commercialized next-generation Sequencing technologies. 1. It potentially reaches long read length >5 kbp with speed 1 bp/ns. 2. Detection of bases is fluorescent tag-free. 3. Except for the use of exonuclease for holding up ssDNA and nucleotide cleavage, an involvement of enzyme is remarkably obviated in nanopore sequencing. This implies that nanopore sequencing is less sensitive to temperature throughout the sequencing reaction and reliable outcome can be maintained. 4. Instead of sequencing DNA during polymerization, single DNA strands are sequenced through the nanopore using DNA strand depolymerization. Hence, hands-on time for sample preparation can be shortened significantly. No cloning and amplification steps required.
The MinION can be used by connecting into a laptop or PC using a high-speed USB 3.0 cable. No additional high end computing infrastructure is required.
Oxford Nanopore MinION flow cell can now generate DNA sequence data from 10 to 20 Gb. It offers Ultra-long read lengths (hundreds of kb) are possible as you can choose your fragment length. This technology designed to streams data in real time, this helps to do analysis during the experiment and workflows.
The Oxford Nanopore MinION is a portable sequencing device that already has proven its power in e.g. the rapid identification of viral pathogens, the monitoring of ebola, genome assembly improvement, and environmental and food safety monitoring.
(16s rRNA / 18s rRNA / COI / RBCL etc.,)