Figure 1

Illustration of PacBio sequence generation. Adaptors (SMRTbells) are first ligated to each amplicon, and after a sequencing primer is annealed to the SMRTbell template, DNA polymerase is bound to the complex. This polymerase-amplicon-adaptor complex is then loaded into zero-mode waveguides (ZMWs) where replication occurs, producing nucleotide-specific fluorescence. Circular consensus sequencing (ccs) allows the polymerase to repeatedly replicate the circularized strand, producing one long read with randomly distributed errors[9]. Post-run, the SMRTbell sequences are bioinformatically trimmed away, single-molecule fragments are aligned, and a consensus sequence is generated. The single-molecule coverage and accuracy of resulting ccs reads are amplicon- and read-length dependent, with smaller amplicons and longer reads giving higher single-molecule coverage and thus higher ccs read accuracy.