Second-generation sequencing

SGS was developed in the early 2000s and became common use in DNA and RNA sequencing experiments around 2010s (the first genome completely sequenced with NGS, in particular 454-sequencing which we will further describe in this section, was published in 2008). 

SGS methods are characterised by different underlying technologies, but they all include the following: 

• DNA fragments have to be amplified through PCR prior to sequencing; this step is carried out on the sequencing slide. This is essential for signal detection
• The reads produced are shorts, with a range from 70 to a few hundred base pairs
• They are all ‘sequencing-by-synthesis’ technologies, with a signal generated for each nucleotide added

SGS technologies

• Illumina (Solexa) sequencing: Illumina sequencing works by simultaneously identifying DNA bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain
• Roche 454 sequencing: This method is based on pyrosequencing, a technique which detects pyrophosphate release, using light signal (bioluminescence), after nucleotides are incorporated by polymerase to a new strand of DNA. Roche 454 sequencing platform has been discontinued since 2016.
• Ion Torrent: Proton / PGM sequencing: Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase and therefore differs from the previous two methods as it does not measure light.

The next few pages provide step-by-step explanations of how each of these technologies work.