2 Base Encoding - How IT Works

How It Works

The SOLiD Sequencing System uses probes with dual base encoding.

The underlying chemistry is summarized in the following steps:

- Step 1, 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 resulting library contains template DNA fragments, which are tagged with one adapter at each end (A1-template-A2).

- Step 2, Emulsion PCR: In this step, the emulsion (droplets of water suspended in oil) PCR reaction is performed using DNA fragments from library, two primers (P1 and P2) that complement to the previously used adapters (P1 with A1 and P2 with A2), other PCR reaction components and 1μm beads coupled with one of the primers (e.g. P1). make dilution from DNA library to maximize the droplet that contain one DNA fragment 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. Dressman et al. first describe this technique in 2003.

- Step 3, Bead Enrichment: In practice, only 30% of beads have target DNA. To increase the number of beads that have target DNA, 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 resulting complex will be separated from untargeted beads, and melt off to dissociate the targeted beads from polystyrene. This step can increase 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.

- Step 4, 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.

- Step 5, 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 are eight bases in length with a free hydroxyl group at the 3’ end, a fluorescent dye at the 5’ end and a cleavage site between the fifth and sixth nucleotide. The first two bases (starting at the 3' end) are complementary to the nucleotides being sequenced. Bases 3 through 5 are degenerate and able to pair with any nucleotides on the template sequence. Bases 6-8 are also degenerate but are cleaved off, along with the fluorescent dye, as the reaction continues. Cleavage of the fluorescent dye and bases 6-8 leaves a free 5' phosphate group ready for further ligation. In this manner positions n+1 and n+2 are correctly base-paired followed by n+6 and n+7 being correctly paired, etc. The composition of bases n+3,n+4 and n+5 remains undetermined until further rounds of the sequencing reaction.

The sequencing step is basically composed of five rounds and each round consists of about 5-7 cycles (Figure 2). Each round begins with the 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 are added and ligated according to their first and second bases. Then, 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 allows the sequencing of about 25 base pairs of the template from P1.

- Step 6, Decoding Data: For decoding the data, which are represented as colors, we must first know two important factors. First, we must know that each color indicates two bases. 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. Therefore, since each color represents two nucleotides in which the second base of each dinucleotide unit constitutes the first base of the following dinucleotide, knowing just one base in the sequence will lead us to interpret the whole sequence(Figure 2).