Bioinformatics Genomics Next Generation Sequencing

Sequencing Methods Used in NGS

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Certain competing methods of Next Generation Sequencing have been developed by different companies.

454 Pyrosequencing

Pyrosequencing is based on the ‘sequencing by synthesis’ principle, where a complementary strand is synthesised in the presence of polymerase enzyme. In contrast to using dideoxynucleotides to terminate chain amplification (as in Sanger sequencing), pyrosequencing instead detects the release of pyrophosphate when nucleotides are added to the DNA chain. It initially uses the emulsion PCR technique to construct the polonies required for sequencing and removes the complementary strand. Next a ssDNA sequencing primer hybridizes to the end of the strand (primer-binding region), then the four different dNTPs are then sequentially made to flow in and out of the wells over the polonies. When the correct dNTP is enzymatically incorporated into the strand, it causes release of pyrophosphate. In the presence of ATP sulfurylase and adenosine, the pyrophosphate is converted into ATP. This ATP molecule is used for luciferase-catalysed conversion of luciferin to oxyluciferin, which produces light that can be detected with a camera. The relative intensity of light is proportional to the amount of base added (i.e. a peak of twice the intensity indicates two identical bases have been added in succession).

Ion torrent semiconductor sequencing

Ion torrent sequencing uses a “sequencing by synthesis” approach, in which a new DNA strand, complementary to the target strand, is synthesized one base at a time. A semiconductor chip detects the hydrogen ions produced during DNA polymerization.

Following polony formation using emulsion PCR, the DNA library fragment is flooded sequentially with each nucleoside triphosphate (dNTP), as in pyrosequencing. The dNTP is then incorporated into the new strand and is complementary to the nucleotide on the target strand. Each time a nucleotide is successfully added, a hydrogen ion is released, and it detected by the sequencer’s pH sensor. As in the pyrosequencing method, if more than one of the same nucleotide is added, the change in pH/signal intensity is correspondingly larger.

Sequencing by ligation (SOLiD)

SOLiD is an enzymatic method of sequencing that uses DNA ligase, an enzyme used widely in biotechnology for its ability to ligate double-stranded DNA strands. Emulsion PCR is used to immobilize/amplify a ssDNA primer-binding region (known as an adapter) which has been conjugated to the target sequence (i.e. the sequence that is to be sequenced) on a bead. These beads are then deposited onto a glass surface, a high density of beads can be achieved which in turn, increases the throughput of the technique.

Once bead deposition has occurred, a primer of length N is hybridized to the adapter, then the beads are exposed to a library of 8-mer probes which have different fluorescent dye at the 5′ end and a hydroxyl group at the 3′ end. Bases 1 and 2 are complementary to the nucleotides to be sequenced while bases 3-5 are degenerate and bases 6-8 are inosine bases. Only a complementary probe will hybridize to the target sequence, adjacent to the primer. DNA ligase is then used to join the 8-mer probe to the primer. A phosphorothioate linkage between bases 5 and 6 allows the fluorescent dye to be cleaved from the fragment using silver ions. This cleavage allows fluorescence to be measured (four different fluorescent dyes are used, all of which have different emission spectra) and also generates a 5’-phosphate group which can undergo further ligation. Once the first round of sequencing is completed, the extension product is melted off and then a second round of sequencing is performed with a primer of length N−1. Many rounds of sequencing using shorter primers each time and measuring the fluorescence ensures that the target is sequenced.

Reversible terminator sequencing (Illumina)

Reversible terminator sequencing differs from the traditional Sanger method in that, instead of terminating the primer extension irreversibly using dideoxynucleotide, modified nucleotides are used in reversible termination. While many other techniques use emulsion PCR to amplify the DNA library fragments, reversible termination uses bridge PCR, improving the efficiency of this stage of the process.

Reversible terminators can be grouped into two categories: 3′-O-blocked reversible terminators and 3′-unblocked reversible terminators.

3′-O-blocked reversible terminators

The mechanism uses a sequencing by synthesis approach, elongating the primer in a stepwise manner. Firstly, the sequencing primers and templates are fixed to a solid support. The support is exposed to each of the four DNA bases, which have a different fluorophore attached (to the nitrogenous base) in addition to a 3’-O-azidomethyl group.

Only the correct base anneals to the target and is subsequently ligated to the primer. The solid support is then imaged and nucleotides that have not been incorporated are washed away and the fluorescent branch is cleaved using TCEP (tris(2-carboxyethyl) phosphine). TCEP also removes the 3’-O-azidomethyl group, regenerating 3’-OH, and the cycle can be repeated.

3′-unblocked reversible terminators

The reversible termination group of 3′-unblocked reversible terminators is linked to both the base and the fluorescence group, which now acts as part of the termination group as well as a reporter. This method differs from the 3′-O-blocked reversible terminators method in three ways: firstly, the 3’-position is not blocked (i.e. the base has free 3’-OH); the fluorophore is the same for all four bases; and each modified base is flowed in sequentially rather than at the same time.

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