After addition of barcode and primer binding sites to the amplicon or genomic fragments, they are denatured into single strands. A splint oligo compatible with the two sequencing primers at the ends joins the ends into a circle for rolling circle amplification of a single strand.

For pair-end sequencing there are 3 primers used:

1. Sequencing in the first direction. Second strand synthesis occurs on the single strand concatemer;

2. Sequencing occurs in the reverse direction;

3. A primer next to the barcode sequences the barcode.