With the LED-TIRF Transformer built and the Sequencing Reservoir consumable assembled, you are ready to begin sequencing!
The first step to sequencing is to prepare a sequencing library. This entails taking your DNA sequence of interest and adding specific known DNA sequences to it. This process allows you to sequence your unknown sequence of interest on the Transformer.
The library molecules will all contain the complement to the surface-bound forward primer on the sequencing substrate. This allows each molecule to hybridize to the sequencing substrate so a polymerase can initiate surface amplified cluster generation.
Library molecules also contain the surface-bound reverse primer sequence. As forward strands are generated using the library molecules as template, the complement of the surface-bound reverse primer is polymerized so the surface-bound reverse primers can anneal and a polymerase can extend reverse strands. As more copies of forward and reverse strands are produced branching off of the surface and the growing cluster, the cluster becomes hyperbranched.
Another important library element is the site for the sequencing primer. The hyperbranched surface amplified clusters will contain a primer binding site just upstream of the DNA sequence of interest. When you’re ready to sequence, the sequencing primer will anneal to this site. The first base extended from this sequencing primer will be the first base of your DNA of interest.