If you follow the latest trend of the sequencing technology, you may notice there is a leap of sequencing quality, volume and cost from Sanger to NGS, making possible of whole genome or transcriptome sequencing for all type of living organisms, including those species which carrying extremely long or complex genomes. The advent in NGS has massively rocketed the field of genomic research as well as many other downstream applications.
Before the 1990s, Sanger sequencing is considered the only method to obtain the reading of a DNA fragment. Sanger sequencing uses specific chain-terminating nucleotides, which is called as dideoxynucleotides that lacking a 3'-OH group. As a result, no phosphodiester bond can be formed by DNA polymerase, leading to the termination of the growing DNA chain at that position. These ddNTPs are added with a radioactive or fluorescent element, enabling the detection in "sequencing" gels or automated sequencing machines, respectively.
The main success of NGS technology is the ability to obtain high throughput, low cost, and better sequence quality. This is because NGS technology allows millions of fragments to be sequenced at the same time in a single run, as compared to Sanger sequencing which only produces one limited replicate. The Sanger method required separate steps for sequencing, separation (by electrophoresis) and detection, which made it difficult to automate the sample preparation and it was limited in throughput, scalability and resolution. The NGS method uses array-based sequencing which combines the techniques developed in Sanger sequencing to process millions of reactions in parallel, resulting in very high speed and throughput at a reduced cost. The genome sequencing projects that took many years with Sanger methods can now be completed in hours with NGS, although with shorter read lengths (the number of bases that are sequenced at a time) and less accuracy.
NGS sequencing technology can be grouped into two major categories, sequencing by hybridization and sequencing by synthesis (SBS). SBS methods are a further development of Sanger sequencing, without the dideoxy terminators, in combination with repeated cycles of synthesis, imaging, and methods to incorporate additional nucleotides in the growing chain.
The earlier version of SBS machines generated much shorter reads (100-300 bases) compared to the classical Sanger method, which also causes difficulty in the assembly process and potential sequence context errors. This is generally named as "short-read sequencing". However, these shortcomings and limitations have been improved gradually in the latter sequencing models.
One can obtain a better sequencing insight by incorporating the data from different sequencing technologies, such as Single Molecule Real Time DNA Sequencing (SMRT) that targeting the generation of long-read data, nanopore sequencing in achieving real-time and ultrafast sequencing results. Anyway, one can look for some comprehensive literature reviews to understand this part better, or I shall make a summary next time, hehe.
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