Schematic representation of SMRT sequencing. DNA polymerase synthesizes a nascent strand complementary to a closed-circular SMRTbell DNA template. Fluorescent phospholinked nucleotides produce real-time fluorescent pulse data for both basecalls and incorporation kinetics. Inter-pulse distance (IPD) is defined as the time gap between two consecutive pulses and includes the time taken to move the DNA polymerase between base positions on the DNA template. Pulse width (PW) is the duration of a given pulse and represents the residence time of the nucleotide at the active site of DNA polymerase, up to the point of incorporation and cleavage of the fluorescent tag. Closed SMRTbell sequencing templates yield multiple, overlapping subreads of both forward and reverse strands of the insert, which are then assembled in silico with the primary consensus core algorithm into circular consensus sequence (CCS), eliminating randomly distributed pulse-read sequencing errors (Supplemental Table S1). Note also that although a single enzymatic misincorporation is indicated for completeness, such errors occur during polymerization with a frequency that is several orders of magnitude less than photonic miscall errors and therefore do not contribute to the error profile.