Roche's 454 technology, illumina's Solexa/Hiseq technology and ABI's SOLID technology mark the birth of the second generation sequencing technology. Roche's 454 sequencing system is the first commercial sequencing platform in the second generation sequencing technology.
Among them, the market size of Illumina accounts for more than 75%, mainly Miseq and Hiseq. Below? This paper mainly introduces the principle of PE(Pair End) sequencing:
Noun:
Flow cell: the carrier/container of sequencing reaction. There are 8 lanes in 1 flow cell.
Lane: the parallel lane of sequencing reaction, where reagent addition and elution are carried out.
Tiling: the position of each fluorescence scan is invisible to the naked eye.
Double-ended sequencing: the sequence may be four or five hundred bp long, with 120- 150bp on both sides.
Junction: some undetected areas in the middle of double-ended sequencing.
Index (bar code): A swimming lane usually needs to measure multiple samples, and each sample is marked with a specific sequence to distinguish different samples.
Flow cell structure: A channel contains two strips with 60 tiles in each column, each tile will be planted with different clusters, and each tile will be photographed four times in a cycle (once for each substrate).
After the interruption, the end will be uneven and filled with enzyme, so the current sequence is blunt.
After leveling is completed, a specific base A is added to the 3' end by enzyme, and after adding A, the principle of complementary pairing can be used. After adding adapters, adapters can be divided into two parts, one is the primer sequence needed for sequencing, and the other is the primer sequence needed for library construction and amplification.
Carry out PCR amplification to make our DNA sample concentration enough to meet the requirements of computer.
Reads 1 and reads2 do not overlap.
Flow cell is a channel for adsorbing flowing DNA fragments, and sequencing is carried out here. The sequence to be tested in the library constructed above is pre-configured with a certain concentration, and when it passes through here, it will strongly and randomly attach to lane under the action of specific chemical reagents, pairing with the short sequence above. As a result of loading, lane adsorbed washed away DNA, and it can be amplified by bridge PCR on the surface.
Forward strand of double-ended sequencing:
Why is Illumina sequencing limited in length?
Hiseq2000 sequencer
The sequencer is equipped with two flow cells, which are called double flow cells. The classic Hiseq2500 can generate 700-800Gb of data at a time (where Gb is the number of sequencing bases, which is different from the number of bytes).
Data amount = single-ended read length * single-ended read times * 2(PE)
Sequencing depth = data size/reference genome size
This is a new milestone. Marked by SMRT of PacBio Company and nano-pore single molecule sequencing technology of Oxford Nano-pore Technology Company, it is called the third generation sequencing technology. Compared with the previous two generations, the biggest feature is single molecule sequencing, which does not need PCR amplification, and the reading length is extremely long, reaching 10Kb- 15Kb on average, which is more than 100 times that of the second generation sequencing technology. It is worth noting that the reading length of these sequences is no longer equal during sequencing.
1./p/ 10 1c 14c 3a 1 D2
2./p/20702684
3./s/tw hwa-f 1 RNP _ xwy 66 p 12pg
4./s/9KUY43lD5miLdPZJKgRV0A