Different Platforms of sequencing ... Part IV
SOLiD Sequencing:
Step 1 : Library Preparation
Preparation of the two types of libraries such as fragment library or mate-paired library.Mate-paired libraries are libraries where two fragments of sequences are jointed with adapter and are known distant apart in the target.
Step 2 : Emulsion PCR/Bead Enrichment
Clonal bead populations in microreactors are prepared with template, PCR reaction components, beads, and primers. After PCR the templates were deanatured and bead enrichment process goes on to separate beads with extended templates from undesired beads. The template on the selected beads undergoes a 3’ modification to allow covalent attachment to the slide.
Deposit 3` modified beads onto a glass slide (Figure 1). During bead loading, deposition chambers enable you to segment a slide into one, four, or eight sections. A key advantage of the system is the ability to accommodate increasing densities of beads per slide, resulting in a higher level of throughput from the same system.
Figure 1 : Attachment of beads in glass slide
Primers hybridize to the P1 adapter sequence on the templated beads (Figure 2).
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.
Figure 2 : Sequencing by Ligation
Five rounds of primer reset are completed for each sequence tag (Figure 3). 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. This dual interrogation is fundamental to the unmatched accuracy characterized by the SOLiD System.
Figure 3 : Primer Reset
