SOLiD® Next-Generation Sequencing Chemistry
- Prepare one of the two types of libraries (Figure 1) for SOLiD® System sequencing—fragment or mate-paired. Your choice of library depends on the research application you're performing and the information you desire from your experiments. Note: paired-end sequencing is performed with fragment libraries.
- Using the automated SOLiD® EZ Bead™ System, clonal bead populations are prepared (Figure 2) in microreactors containing template, PCR reaction components, beads, and primers.
- Beads with extended templates are enriched on the SOLID® EZ Bead™ System and the template on the selected beads undergoes a 3’ modification to allow covalent attachment to the slide or FlowChip.
- Deposit 3' modified beads onto a glass slide or FlowChip(s) (Figure 3).
Sequencing by Ligation
- Primers hybridize to the P1 adapter sequence on the templated beads (Figure 4).
- A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction.
- Multiple cycles of ligation, detection, and cleavage are performed with the number of cycles determining the eventual read length.
- Following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n–1 position for a second round of ligation cycles.
- Five rounds of primer reset are completed for each sequence tag (Figure 5). Through the primer reset process, virtually every base is interrogated in two independent ligation reactions by two different primers.
For example, the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1 (see figure at right). This dual interrogation is fundamental to the unmatched accuracy characterized by the SOLiD® System.
Exact Call Chemistry
- Sequencing with an additional primer using a multi-base encoding scheme is performed using the Exact Call Chemistry (ECC) module. In combination with a reference, up to 99.99% accuracy is achieved.
Using the ECC module without a reference enables data output in base space.