2 base encoding
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Introduction
The dream of human whole genome re-sequencing at a reasonable time and cost (less than $1000) is coming to be true with recently developed next-generation sequencing technologies. These technologies generating hundreds of thousands of small sequence reads at one time include mainly the 454 pyrosequencing (introduced 2005), Solexa system (introduced 2006) and 2-base encoding sequencing (introduced 2007-2008). These methods have reduced the cost from almost 0.01 $/base in 2004 to near 0.0001 $/base in 2006 and increased the sequencing machine capacity from 1,000,000 base/machine/day in 2004 to more than 100,000,000 base/machine/day in 2006. The general steps in all of these next-generation sequencing techniques include:
1- Random fragmentation of genomic DNA
2- Immobilization of single DNA fragments on a solid support like a bead or planar solid surface
3- Amplification of DNA fragments on the solid surface using PCR and making polymerase colonies
4- Sequencing and subsequent in situ interrogation after each cycle using fluorescence scanning or chemiluminescence [1].
In 2005 Shendure et al. used a sequencing procedure using multiple cycles of ligation of fluorescent labled 9-mer probes which distinguish the central base. In each cycle the sequence of every fifth base is recognized. This process is repeated using different primers to sequence the remaining four bases in each gap [2]. The most recent next-generation sequencing technology which is called 2-base encoding or SOLiD (Sequencing by Oligonucleotide Ligation and Detection) technology has been developed by Applied Biosystem and will be commercially available in 2008. Similar to Shendure et al. and despite other two next-generation sequencing technologies, 2-base encoding is based on ligation sequencing rather than sequencing by synthesis. However, its fundamental difference to previously used 9-mer probes with distinguished central base is taking advantage of fluorescent labeled 8-mer probes with distinguished the 2 central bases.
This review will take a brief overview on the 2-base encoding technology and will compare it to the other next-generation sequencing technologies.
How it works
The SOLiD Sequencing System uses probes with dual base encoding.
For decoding the colors we must first know that each single color indicates two bases and second, we need to know one of the bases in the sequence[3].
Advantages
Detection of SNP
Detection of errors
Limitations
Conclusion
| Next-generation
sequencing method |
Number of
bp/run |
Duration of
each run |
Read length
(bp) |
Cost | Accuracy |
|---|---|---|---|---|---|
| 454 | 100 million | 7.5 hours | up to 250 | 1:10-20 of
Sanger sequencing |
> 99.5% |
| Solexa (1G) | 1000 million | 3 days | up to 50 | 1:20-30 of
454 sequencing |
> 99.93% |
| 2 base encoding | 2000 million | 4 days | up to 35 | 1:20-30 of
454 sequencing |
> 99.94% |
References
- ^ http://www.blackwell-synergy.com/doi/full/10.1111/j.1471-8286.2007.02019.x?cookieSet=1 Sequencing breakthroughs for genomic ecology and evolutionary biology
- ^ http://www.sciencemag.org/cgi/content/full/309/5741/1728 Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome
- ^ http://seqanswers.com/forums/showthread.php?t=10