Solid Technology
Solid sequencing technology was put into commercial sequencing application by ABI company in 2007. It is based on ligase-mediated method, which is to sequence in the DNA ligation process.
Its principle is:
A. Construction of DNA Library
Add specific adapters to both ends of interrupted DNA fragments, and ligate them into vectors to construct a single stranded DNA library.
B. Emulsion PCR
PCR process of Solid is similar to the 454 method, which also uses emulsion PCR. But these micro drops are much smaller than those of 454 system, which are only 1um in size. The 3 'end of the amplified product is modified at the time of amplification, which is prepared for the next step of sequencing process. Modified micro drops will be deposited on a glass slide before loading. The biggest advantage of Solid system is that each slide can accommodate micro drops with much higher density than 454, which can easily achieve higher throughput in the same system.
C. Ligase Sequencing
This step is unique to Solid sequencing. It does not rely on the previously and commonly used DNA polymerase in sequencing, but on the ligase. The substrate of the Solid ligation reaction is a mixture of 8-bases single stranded fluorescent probes, which are simply represented as 3 '-XXnnnzzz-5'. These probes are paired with single strand DNA templates according to base complementary rules. The 5’ ends of these probes are labeled by CY5, Texas Red, CY3 and 6-FAM, which are 4 kinds of fluorescent dyes. For the 8-bases single stranded fluorescent probe, the first two bases are confirmed, and different fluorescent markers are added to the latter three bases (zzz) according to the probes sorts. Two bases determine a fluorescent signal, which means to confirm two bases at a time. This sequencing method is also called two-base sequencing. When the fluorescent probe can be paired with the DNA template, it will emit a fluorescence signal representing the first and second bases. After recording the fluorescence signal, the chemical method is used to cut the bond between the fifth and sixth base, so that the fluorescence group can be removed for the next step. But it is worth noting that, through this sequencing method, there are 5 bases gap between two confirmed sites. In the end of first sequencing cycle, the new synthesized strand would go through denaturation and elution. Then the second round of sequencing is going by universal sequencing primer at the n-1 site. The universal primer n-1 and primer n have a site difference in pairing position, which means the primer n-1 would pair with the adapter from the n-1 site. That is, through the primer n-1, sequencing result would initiate from the n-1 site, then the first, fifth, and sixth site of template sequence. Then, using n-2, n-3 and n-4 primers to repeat above process, until the fifth round of sequencing, the template sequencing can be completed at all sites, and each site would be detected for two times. The read length of this technique is 2×50bp, and the subsequent sequence stitching is also complicated. Due to the twice detection for one site, the original sequencing accuracy of this technology is as high as 99.94%, while the accuracy of 15x coverage could be up to 99.999%. It should be said that in current generation of sequencing technology, it has the highest accuracy. But in the fluorescence decoding stage, it is easy to result in a series of decoding error, for the fluorescence signal is determined by the former two bases.
References:
- Mardis, E. R. Next-generation DNA sequencing methods. Annual review of genomics and human genetics 9, 387–402 (2008).
- 2. Metzker, M. L. Sequencing technologies - the next generation. Nature reviews. Genetics 11, 31–46 (2010).
Related Links