Next – Generation Sequencing

The Applied Biosystems sequencers includes Ion Torrent, Ion S5, Ion Proton and Ion PGM systems and it use a pH modification to detect a base change. The voltage modification is recorded once a ion is discharged upon addition of a nucleotide base is known as semiconductor sequencing. In this kind of sequencing there is no need for the alteration of nucleotides or the addition of associate enzyme cascade for detection.

Ion Torrent is one of the primary semiconductor sequencing chip accessible on the industrial market. Rather than pyrophosphate detection, it performs sequencing by detecting hydrogen ions. the fundamental methodology of ion Torrent is that the same as that of Roche 454. It conjointly uses emPCR to amplify the templates and therefore the amplified beads are loaded into a high-density array of wells, and every well contains one template. In every iteration, one type of dNTP flows across the wells. If the dNTP is complementary to the template, enzyme can extend by one base and release H+. the comfort of H+ modifications the pH within the well and device at all-time low of the well measures the pH change and converts it into electrical signals. The device avoids the utilization of optical measurements, that need an advanced camera and optical maser. this is often the most distinction between ion Torrent sequencing and 454 sequencing. The unattached dNTP molecules are washed out before consequent iteration. By deciphering the flow-gram obtained from the device, we are able to recover the sequences of the templates. Since the strategy employed by Ion Torrent is analogous thereto of Roche 454, it conjointly has the disadvantage that it cannot distinguish long homopolymers.

Sequencing by tying Platforms – SOLiD

Applied Biosystems SOLiD instrument employs a ligation and detection approach. The sequencing is performed by DNA ligase rather than DNA enzyme. The extracted DNA is fragmented by restriction enzymes, and P1 and P2 adaptors are added by PCR. The fragments are annealed to clonal emulsion beads via the universal P1 adapter.

Instead of extending the template base by base polymerase enzyme, ligase mediated strategies use probes to examine the bases on the template. ABI SOLiD is that the industrial sequencer that uses this approach. In SOLiD, the templates are 1st amplified by emPCR. Adding to that, millions of templates are placed on a plate and then SOLiD technology tries to probe the bases of all templates in parallel. In each iteration, SOLiD probes 2 adjacent bases of every template, i.e., it uses two-base color cryptography. the first advantage of the two-base color cryptography is that it improves the only nucleotide variation detection line of work. Since each base is roofed by 2 color bases, it reduces the error rate.

Roche 454

It can do the incorporation of a deoxyribonucleotide triphosphate (dNTP) into a growing DNA strand involves the formation of a covalent bond and therefore the unleash of pyrophosphate and a charged cation. Hence, it’s attainable to sequence the DNA model by ion detection concentration modification or pyrophosphate modification. Roche 454 and ion Torrent are 2 sequencers that get profited by this principle. The Roche 454 sequencer performs sequencing by detecting pyrophosphates and as known as pyrosequencing. Intially, 454 sequencer adopt emPCR to amplify the templates. Then, amplified beads are loaded into associate array of wells. Here, each well contains one amplified bead that corresponds to at least one DNA template. In every iteration, one style of dNTP flows across the wells. If the dNTP is complementary to the model in a very well, enzyme can extend by one base and relax pyrophosphate. With the assistance of enzymes sulfurylase and luciferase, the pyrophosphate is reborn into visual light. The CDC camera detects the signal from all wells in parallel. for every well, the light intensity generated is recorded as a flowgram.