There are several sequencing technologies that are prevalent, along with some that are becoming more common.
Sanger sequencing, the first DNA sequencing technology, is a sequencing-by-synthesis technique employing chain-terminating dideoxy nucleotides (ddNTPs). Fluorescent ddNTPs can be used when capillary gel electrophoresis is used, allowing fully automatic sequencing. This technique is highly accurate, and has long read lengths of around 800 base pairs.
Illumina sequencing is a second generation sequencing technology, carrying out thousands of simultaneous Sanger sequencing reactions. By using reversible-terminator fluorescent nucleotides, the DNA strand can be extended one base at a time, allowing the fluorescence (or nucleotide) to be identified at each position. Bridge amplification is used to form clusters of identical DNA strands by PCR. Using a restriction enzyme, a restriction site in the adaptor molecules can be cleaved, ensuring all DNA strands are in the same direction (5’→3’ / 3’→5’). The DNA is bound to DNA probes immobilised on a flow cell. This has a high accuracy due to the consensus sequence produced, but a shorter read length of around 150 base pairs. To form a complete sequence, the short sequences must be assembled into a long sequence read, in the same way shotgun sequencing does. This can take time, especially with large genomes.
The most recent developments in DNA sequencing are PacBio and Oxford Nanopore. PacBio uses fluorescent nucleotides with a DNA polymerase immobilised in the base of a zero-mode waveguide (ZMW). The ZMW is a small well in the surface of a slide, with a diameter approximately the same as the wavelength of the light being used to illuminate it. This allows only the nucleotide being incorporated into the DNA strand to be illuminated, which can be recorded by an optical sensor. PacBio sequencing also captures the methylation state of the DNA, based on the time taken to incorporate new nucleotides. A methylated cytosine causes a slight pause before the next nucleotide is incorporated. Although this technology has a slightly lower accuracy that Illumina sequencing, consensus sequences can easily be obtained by using PacBio SMRTBell adaptors. These produce a circular DNA molecule from linear dsDNA, allowing the DNA strand to pass through the polymerase several times. Read lengths are up to 50kb.
Nanopore sequencing uses a pore protein (CsgG) embedded in an electrically insulating artificial membrane. The DNA or RNA is passed through this pore by a motor protein (likely a helicase), and the electron flow around the nucleic acid strand is recording. This can then be converted to a signature, allowing conversion to a nucleotide sequence. Like PacBio, Nanopore sequencing can also provide data on methylation of the DNA, as it has a different electron signature. Nanopore has very long read lengths, up to 2 Mb, and does not require library preparation. This reduces the likelihood of discontinuous sequences being obtained, overcoming repeat regions.
