A Simple Comparison of Sequencing Techniques
Posted by Debbie Evans on February 16th, 2016
The first generation sequencing technology: Sanger Sequencing
In amplification process, we use 4 different marked double terminal deoxy bases to terminate the chain extension, and then to separate different length of the amplified fragments by electrophoresis. According to the chain-end-fluorescent marker to identify what is the end base of different length of chain, to know this end base location and category, we have all known sites to connect and make clear the sequence of the whole chain.
The reason for still being used:
Length of sequence (700-1000nt)
Low error rate
High single base quality
Disadvantages: sequencing flux is low (generally 96 holes).
The second generation sequencing technology:
The second generation sequencing is also known as deep sequencing, large-scale parallel sequencing, etc. There are 4 main platforms:
454 FLX：Pyrosequencing, detection of pyrophosphate, emulsion PCR;
Genome Analyzer: Sequencing-by-synthesis with reversible terminators, bridge PCR;
SOLiD: Sequencing by ligation, emulsion PCR;
Ion Torrent: Pyrosequencing, detection of proton potential, emulsion PCR.
454 FLX
In 2005, it was the first second generation sequencing technology.
Without electrophoresis, sequencing and synthesis at the same time;
DNA fragments also need not fluorescent labeling;
When base is added to the sequence, one pyrophosphate is took off. Through the detection of pyrophosphate to know what base is added.
HiSeq 2000/2500
Now it is the most common sequencing technology. Its sequencing principle is the same as that of Analyzer Genome by using the stable reversible termination method to synthesize and sequence simultaneously. The technique uses four contained terminal block groups and different fluorescence signal bases for complementary template strand synthesis, not only to ensure the high precision and high order of the sequencing, but also eliminate sequencing errors caused by the repetitive sequences and homopolymer.
Ion Torrent
Ion Torrent platform is a high throughput sequencing instrument based on semiconductor technology. The platform uses a high density semiconductor chip covered with small holes. A small hole is a sequencing reaction tank, with a sensor at the bottom of the hole. Compared with other new generation sequencers, ion torrent sequencer does not require excitation light, CCD imager and fluorescent labeling. It can directly and fast "read" DNA sequences so that it has significant advantages of simplicity, accuracy, flexibility and low cost.
