Suzanne de Bruijn

45 The impact of modern technologies on molecular diagnostics A Library preparation B Sequencing process High quality DNA strand Ligate adapters and size select Anneal primers and bind DNA polymerase SMRT-bell Circular consensus sequence (CCS) or HiFi read (>99% accuracy) Load library into SMRT cell SMRT cell (top view) Trimmed adapters from reads to yield subreads Generate consensus read Single ZMW (cross section) Figure 1. Overview of SMRT sequencing. (A) Sequencing starts with library preparation of ultra-long dsDNA. In the next step, adapters, DNA polymerase, and primers bind to the dsDNA creating the SMRT-bell, which later will be loaded to the SMRT-cell. (B) The library is randomly distributed in the SMRT-cell in the sequencer instrument, in the ideal condition 1/3 of the ZMWs will be loaded by an SMRT-bell. In each ZMW, DNA polymerase together with an SMRT-bell are bound to the bottom of the ZMW. The SMRT sequencing uses the circular DNA template to generate a continuous long read in each ZMW chamber. Afterwards, the adapters are trimmed from this long read and overlapping reads can be combined to one consensus sequence of high quality HiFi read.