DNaseI and flowcell clearing for increasing long read yields and multi-sample sequencingThe use of the Nuclease flush protocol (DNaseI digestion of DNA on the surface of the flowcell) has become an integral part of our long reads sequencing efforts. These can be seen in the figure above (Protocol 'Modified LSK109 ligation prep with needle shear and bead clean up') and can be seen as increases in data yield accumulations on the curves. In the case of our native rat genomic sequencing we see an accumulation of pore blockage over time that is DNA fragment length dependent. Shown below are two libraries produced from an identical sample sheared to an increasingly smaller size and run.Using DNaseI surface clearing we often see recovery of an additional 50 \u2013 80 % of total reported pores on top of what a mux scan reports before treatment as long as the flowcell surface has not been \u201cdamaged\u201d in some other way. You can monitor the process live if you restart a run and then pipette in the DNaseI clearing solution and watch the duty plots (see below). Once complete in ~30 mins flushing back to FLB\/PFB and re-tethering will on a restart produce a pore count of what you have left to use. The flowcell is \u201creset\u201d ready for the next library addition and run restart. That may be the same library again or something different\u2026\u2026With our \u201cblocky\u201d rat genomic samples sheared to 18-34kb we have been performing these resets every 16 \u2013 24 hrs. For larger fragment lengths we will likely shorten this time period so the flowcell is not sitting there in an unproductive \u201cblocked\u201d state. It might be time for some MinKNOW scripted automated yield monitoring and hardware surface clearing hacks ;o).