Sequencing by Ligation

Once loaded onto the Analyzer, primers hybridize to the adapter sequence within the library template. A set of four fluorescently labeled di-base probes competes to ligate 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 resets are completed for each sequence tag.

More about how it works:

Principles of Di-Base Sequencing and Color Space Analysis

First Cycle

Second Cycle

Reset

First Cycle (Reset 1)

Mulitple Rounds of Sequencing- Fragment Library

Multiple Rounds of Sequencing- Mate-Pair Library