Amplification-free targeted sequencing on the PacBio platform (PacBio No-Amp). DNA is dephosphorylated to prevent sequencing adapter ligation. Cas9 RNPs with guides specific to the ROI are used to cleave the DNA upstream and downstream from the target(s). Sequencing adapters are ligated to the cleaved products. Since no background reduction has been performed yet, nontarget strands protected by Cas9 on both ends will also likely end up with SMRTbell adapters. Exonucleases are introduced to digest the rest of the background DNA. Only those fragments with SMRTbells attached on both sides survive the exonuclease digestion and make up the sequencing pool that is loaded on to PacBio flowcells. (Created with BioRender; https://www.biorender.com/)
