gDNA seq of Skeletonema costatum PacBio
The first step in the generation of a SMRTbell library is production of appropriately-sized double-stranded DNA fragments. These fragments can be generated by random shearing of DNA, or by amplification of target regions of interest. The SMRTbell library itself is produced by ligating universal hairpin adapters onto double-stranded DNA fragments. The hairpin dimers formed during this process are removed at the end of the protocol using a magnetic bead purification step with size-selective conditions. Adapter dimers are also efficiently removed using PacBios MagBead kit. The final step of the protocol is to remove failed ligation products through the use of exonucleases. After the exonuclease and AMPure PB purification steps, sequencing primer is annealed to the SMRTbell templates, followed by binding of the sequence polymerase to the annealed templates.