2 base encoding

The dream of human whole genome re-sequencing at a reasonable time and cost (less than $1000) is becoming realized with recently developed next-generation sequencing technologies. These technologies generate hundreds of thousands of small sequence reads at one time. Well-known examples include 454 pyrosequencing (introduced 2005), Solexa system (introduced 2006) and the 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.

General features

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 .

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 . 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.

How it works

File:Database-2 base encoding.png

Figure 1- Schematic feature of four different probes used in 2 base encoding.

The SOLiD Sequencing System uses probes with dual base encoding.

The underlying chemistry is summarized in the following steps:

- Step1, preparing a library: This step begins with shearing the genomic DNA into small fragments. Then two different adapters are added (for example A1 and A2). The resultant library contains template DNA fragments, which are tagged with one adapter at each end.

- Step2, emulsion PCR: in this step the emulsion (water in oil emulsion) PCR reaction is performed using DNA fragments from library, two primers (P1 and P2) complement to the previously used adapters (P1 with A1 and P2 with A2), other PCR reaction components and beads coupled with one of the primers (e.g. P1). The aim is locating one DNA template and one bead into a single emulsion droplet.

In each droplet, DNA template anneals to the P1-coupled bead from its A1 side. Then DNA polymerase will extend from P1 to make the complementary sequence, which eventually results in a bead enriched with PCR products from a single template. After PCR reaction, templates are denatured and disassociate from the beads.

File:2 Base Encoding.png

Figure 2- Schematic feature of how 2-base encoding system works. Each base pair in the sequence is read twice and enables this system to minimizes the rate of errors.

- Step3, bead enrichment: in practice, only 30% of beads have target DNA. To enrich the number of these beads, large polystyrene beads coated with A2 are added to the solution. Thus, any bead containing the extended products will bind polystyrene bead through its P2 end. The resultant complex will be separated from untargeted beads, and melt of to dissociate the targeted beads from polystyrene. This step can increases the throughput of this system from 30% before enrichment to 80% after enrichment.

After enrichment, the 3’-end of products (P2 end) will be modified which makes them capable of covalent bonding in the next step. Therefore, the products of this step are DNA-coupled beads with 3’-modification of each DNA strand.

- Step4, bead deposition: in this step, products of the last step are deposited onto a glass slide. Beads attach to the glass surface randomly through covalent bonds of the 3’-modified beads and the glass.

- Step5, sequencing reaction: as mentioned earlier, unlike other next-generation methods which perform sequencing through synthesis, 2-base encoding is based on sequencing by ligation. The ligation is performed using specific 8-mer probes:

These probes consist of eight bases (Figure 1) with a ligation site at the 3’ end, a fluorescent dye at the 5’ end and a cleavage site between the fifth and sixth nucleotide; the first three nucleotides are degenerate bases (N) which can be any of the four A, G, T, C bases. The last three nucleotides (Z) are universal bases which can pair with any of the four A, G, T, C bases. The remaining two bases, fourth and fifth, are the basis of 2-base encoding. The combination of these adjacent dinucleotides will make sixteen different probes which according to limitations of fluorescent dyes are classified into four groups (Figure 1). Therefore, each fluorescent dye represents a class of four different probes. In general, calculating all the possibilities, there will be 1024 octamer probes, 4 dyes each representing 4 nucleotides, and 256 probes per dye.

The sequencing step is basically composed of five rounds and each round consists of about 5-7 cycles (Figure 2). Each round begins with addition of a P1-complementary universal primer. This primer has for example n nucleotides and its 5’-end matches exactly with the 3’-end of the P1. In each cycle, 8-mer probes (1024 probes) are added and ligated according to their fourth and fifth bases. The remaining unbound probes are washed out, the fluorescent signal from the bound probe is measured, and the bound probe is cleaved between its fifth and sixth nucleotide. Finally the primer and probes are all reset for the next round.

In the next round a new universal primer anneals the position n-1 (its 5’-end matches to the base exactly before the 3’-end of the P1) and the subsequent cycles are repeated similar to the first round. The remaining three rounds will be performed with new universal primers annealing positions n-2, n-3 and n-4 relative to the 3'-end of P1.

A complete reaction of five rounds allow sequencing of about 25 base pairs of the template from P1.

- Step6, decoding data: for decoding the data which are represented as colors, we must first know two important factors: first, that each single color indicates two bases and second, we need to know one of the bases in the sequence; this base is incorporated in the sequence in the last (fifth) round of step5. This known base is the last nucleotide of the 3’-end of the known P1 (Figure 2). Therefore, as each color represents two nucleotides in which the second base of each dinucleotide unit constitute the first base of the following dinucleotide, knowing just one base in the sequence will lead us to interpret the whole sequence.

Advantages

-Accuracy: each base in this sequencing method is read and interpreted twice. This can increase the accuracy of the system to more than 99.94% which is higher than other systems.

-Detection of SNPs and other small changes: One of the main advantages of this system is its ease in detection of single nucleotide polymorphisms (SNPs) as well as other small alterations in the template sequence. Changing the color of two adjacent bases is characteristic for SNPs. The detection of other alterations is summarized in Figure 2.

- Detection of errors: as discussed earlier, each base in this system is recognized by two colors and alteration in any single base will result in change in two colors. Therefore, while alteration in two or more than two colors demonstrate a real change in our sequence, just one color change indicates an error and should not be considered as a change (Figure 2).

Conclusion

Some of the features of 2-base encoding are compared to other next-generation sequencing technologies:

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%

Although its disadvantages are still not known, higher throughput, higher accuracy and lower cost of 2-base encoding rather than other methods, make this technology as a promising approach for human whole genome sequencing in the near future.

External Links

• http://www.454.com
• http://www.solexa.com